# Mechanism of homology search expansion during recombinational DNA break repair in Saccharomyces cerevisiae

Agnès Dumont, Nicolas Mendiboure, Jérôme Savocco, Loqmen Anani, Pierrick Moreau, Agnès Thierry, Laurent Modolo, Daniel Jost, Aurèle Piazza

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Agnès Dumont, Nicolas Mendiboure, Jérôme Savocco, Loqmen Anani, Pierrick Moreau, et al.. Mechanism of homology search expansion during recombinational DNA break repair in Saccharomyces cerevisiae. Molecular Cell, 2024, 84 (17), pp.3237-3253.e6. 10.1016/j.molcel.2024.08.003. hal-04887128v2

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Mechanism of homology search expansion during recombinational DNA break repair

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Author list and affiliations

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Agnès Dumont¹, Nicolas Mendiboure¹, Jérôme Savocco¹, Loqmen Anani¹, Pierrick Moreau², Agnès Thierry², Laurent Modolo¹, Daniel Jost¹, Aurèle Piazza¹.*

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¹ Université de Lyon, ENS de Lyon, Université Claude Bernard, CNRS UMR5239, Laboratoire de Biologie et Modélisation de la Cellule, 46 Allée d'Italie, 69007 Lyon, France

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² Unité Régulation spatiale des génomes, Institut Pasteur, CNRS UMR3525, 75015 Paris, France

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* Corresponding Author and Lead Contact: aurele.piazza@ens-lyon.fr

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1 **Summary**

2 Homology search is a central step of DNA double-strand break (DSB) repair by homologous
3 recombination. How it operates in cells remains elusive. We developed a Hi-C-based methodology to
4 map single-stranded DNA (ssDNA) contacts genome-wide in S. cerevisiae, which revealed two main
5 homology search phases. Initial search conducted by short Rad51-ssDNA nucleoprotein filaments
6 (NPFs) is confined in cis by cohesin-mediated chromatin loop folding. Progressive growth of stiff
7 NPFs enables exploration of distant genomic sites. Long-range resection drives this transition from
8 local to genome-wide search by increasing the probability of assembling extensive NPFs. DSB end-
9 tethering promotes coordinated search by opposite NPFs. Finally, an autonomous genetic element on
10 chromosome III engages the NPF, which stimulates homology search in its vicinity. This work reveals
11 the mechanism of the progressive expansion of homology search orchestrated by chromatin organizers,
12 long-range resection, end-tethering, specialized genetic elements, and that exploits the stiff NPF
13 structure conferred by Rad51 oligomerization.
## 1 Introduction

Templated DNA double-strand break (DSB) repair by homologous recombination (HR) entails a search for an intact homologous dsDNA template throughout the genome and the nucleus. This search is conducted by a nucleoprotein filament (NPF) composed of RecA/Rad51 and additional proteins assembled along a hundreds- to kilobases-long ssDNA guide generated by the exonucleolytic resection of each DSB end ¹. The molecular mechanism of dsDNA sampling, defined for bacterial RecA-ssDNA NPFs with *in vitro* single molecule experiments ², occurs at the level of a few RecA monomers and is multiplexed along the length of a single NPF ³,⁴, which can result in multiple independent DNA strand invasion events ⁵⁻⁷. Biochemical, physical and genetic evidence indicate that this inter-segmental search mode extends to eukaryotic Rad51-ssDNA NPFs ⁸⁻¹³.

A conserved property of RecA/Rad51-ssDNA NPFs is to adopt an elongated structure *in vitro*, with persistent length exceeding that of ssDNA by two to three orders of magnitude ¹⁴⁻¹⁷. Recent cytological observations with functional or recessive fluorescently-labeled RecA/Rad51 proteins supported the existence of similar, micrometer-long, stiff fiber-like structures in cells, which led to diverse homology search models in bacteria and eukaryotes ¹⁸⁻²¹. In *S. cerevisiae*, dynamic elongation and collapse of Rad51 structures were proposed to optimize exploration of a spherical nucleus ²⁰. Here we sought to complement these cytological approaches by providing a population-averaged view of genomic sites engaged over time along unmodified Rad51-ssDNA NPFs on both sides of the DSB in *S. cerevisiae*.

Technical limitations in detecting intermediates of homology sampling hampered progress in defining the homology search mechanism. Chromatin immunoprecipitation (ChIP) of Rad51 upon HO-induced DSB formation at the *MAT* locus on chr. III revealed homology-independent enrichment at the chromosome extremities, which was stimulated in a mating-type specific manner by the *cis*-acting recombination enhancer (RE) element ²²⁻²⁴. However, this method (i) precluded studying the role of factors affecting the formation and stability of the NPF, otherwise ablating the experimental readout. Furthermore, it could not resolve which segments of the Rad51-ssDNA NPF contacted other genomic sites. Differently, we recently leveraged Hi-C contact data of dsDNA regions near the DSB site to report on homology search by flanking NPF ²⁵, akin to DSB-flanking fluorescent operator-repressor arrays used to study the localization, association and mobility of DSB ends by cytological approaches ²⁶⁻³⁰. It revealed Rad51- and homology-dependent side-specific contacts between the DSB-flanking dsDNA and an inter-chromosomal donor site, confirming detection of bona fide recombinational interactions. However, this readout remained indirect and may not accurately reflect contacts made by NPF regions distant from the resection front.
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Here we overcame these limitations by developing a Hi-C-based methodology that directly detects contacts made by ssDNA molecules. We uncover the gradual expansion of homology search from local to genome-wide and identify factors involved in regulating this transition.

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# Results

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## A Hi-C-based methodology for ssDNA contacts detection

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Mapping of ssDNA contacts was undertaken using a well-characterized, unrepairable site-specific HO-based DSB induction system in *S. cerevisiae*<sup>31,32</sup> (**Figure 1A**). HO expression is induced upon S-release of G1-synchronized *MAT*a-inc cells, yielding a highly homogeneous population of checkpoint-arrested cells attempting DSB repair by HR in G2/M at 2 and 4h post-DSB induction (**Figure 1B**). The efficient DSB formation in this system (>95% molecules cut in 1h) enables kinetics analysis of the downstream HR steps up to D-loop extension by the left DSB end<sup>25,31,32</sup>. To determine the loci contacted by the DSB-surrounding regions, we employed an optimized Hi-C protocol that yields high-quality stereotypic contacts maps at sub-kilobase resolution (see ref. <sup>33</sup> and **Methods**).

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DSB resection produces long 3'-protruding ssDNA that eliminates restriction sites and the Hi-C readout along the region (**Figure 1C**). To restore, isolate and enrich genome-wide contacts made by ssDNA, we devised a ssDNA-specific Hi-C procedure (hereafter ssHi-C) that relies on DpnII sites restoration upon annealing of 80mer oligonucleotides to the resected ssDNA (**Figure 1D**, **S1A** and **Methods**). These "annealing" oligonucleotides contain single-nucleotide polymorphisms (SNPs), some of which inactivate a secondary restriction site (MfeI or SspI). These secondary restriction sites allow distinguishing contacts made at that site in the ssDNA or dsDNA form (**Figure 1D** and **S1A-B**).

The Hi-C library is then subjected to two rounds of targeted enrichment for the ssDNA sites of interest using biotinylated "capture" oligonucleotides and MfeI/SspI digestion prior to sequencing to eliminate contacts made by these sites in a dsDNA form (**Figure 1D** and **S1A**). An additional SNP-based read filtering step further ensures isolation of ssDNA-specific contacts post-sequencing (**Figure S1B**, **Methods**).

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We designed 21 annealing oligos targeting 19 DpnII restriction fragments on both sides of, and up to 17 kb away from, the DSB site. Out of the 19 ssDNA fragments, 17 are targeted by capture oligos, as well as 7 dsDNA sites used as positive enrichment controls and points of calibration between samples (**Figure 1E**). The two ssDNA fragments not captured are used as negative enrichment controls. One control dsDNA site (ARG4) is at the same distance from a centromere as the DSB site (**Figure S1C**). Annealing oligos partially restored Hi-C coverage, which could be specifically enriched using capture

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oligos (Figure S2A-C). No recovery and enrichment were observed at these sites in the absence of a DSB-inducible construct (Figure S2A-D), indicating that ssHi-C contacts are produced by annealing to a cognate ssDNA. Site-to-site differences in ssDNA fragment enrichment partially depended on the fraction of resected DNA: the greater the resected fraction, the greater the ssDNA enrichment (Figure S2D). Some sites remained poorly restored, despite being fully resected (e.g. L1560 vs L2599, Figure S2B), suggesting site-specific differences in annealing efficiency. Various annealing oligo dropouts indicated that they (i) are each required, (ii) act independently, and (iii) do not interfere with one another for ssDNA site recovery (Figure S2A, C). In conclusion, ssHi-C recovers ssDNA-specific contacts for 16 out of the 17 fragments, with varying site-to-site efficiency.

Despite the strong enrichment for the 16 ssDNA and 7 dsDNA fragments targeted by the capture oligos, 87% of the total contacts originated from the non-targeted fragments. The resulting contact data were highly similar to that of conventional Hi-C (Figure 1F and S2F-H). The 7 control dsDNA sites recapitulated conventional Hi-C contacts, indicating that the capture procedure does not introduce bias at target and non-target sites (Figure S2I-J). ssHi-C libraries can thus be used to study both ssDNA contacts at the enriched sites as well as genome-wide dsDNA contacts classically detected with Hi-C (Figure 1G).

## The genome-wide distribution of ssDNA contacts is not uniform and departs from that of dsDNA in WT cells

Discrete, non-uniform stripes of contacts emanating from the DSB region stretched across chr. V and the genome (Figure 1F and S2F). No such stripes were observed at the 7 captured dsDNA regions, indicating that this contact behavior is specific to ssDNA sites and not an enrichment artefact (Figure 1F). A finer 4C-like representation reveals the propensity of ssDNA on both sides of the DSB to contact inter-chromosomal loci (hereafter *trans* contacts, 64% on average at 4h; Figure 2A). The frequency of ssDNA *trans* contacts increased significantly from 2 to 4h, an opposite trend to the chromatin condensation taking place concomitantly genome-wide^25 (Figure 2B). This *trans* contact frequency was significantly higher than that of (i) average dsDNA Hi-C *trans* contacts (19%), (ii) DSB-flanking dsDNA Hi-C *trans* contacts (33.7%), and (iii) a control captured dsDNA site located at the same distance from a centromere as the DSB site (ARG4, 22%; Figure 2A-B and S3A-B). The distribution of ssDNA-specific *trans* contacts was not uniform over the genome, with local enrichment at centromeres, the donor site, and on chr. III (Figure 2A and see below). Finally, the distribution of *trans* contacts was not uniform along ssDNA, from ~74% for DSB-proximal sites to ~40% for DSB-distal sites 4h post-DSB induction (Figure 2C). Consequently, the predominant ssDNA contacts with
chromatin in *trans* is a decreasing function of the genomic distance to the DSB in wild-type cells.

These observations show that chromatin engagement by ssDNA is not uniform over the genome, along

ssDNA, and over time. DSB-proximal and DSB-distal sites are apart in the nucleus.

4 **DSB-proximal ssDNA contacts with chromatin in cis are broadly distributed**

The distribution of *cis* contacts made by ssDNA sites departed from that of flanking dsDNA regions

detected with Hi-C (Figure 2A):

• ssDNA end similarly contacted chromatin on both sides of the DSB, unlike dsDNA (Figure

S3C).

• ssDNA contacts enrichment with chromatin in *cis* decreased from 2 to 4h, coincident with an

increase in *trans* contact (Figure 2B-C).

• The decay of ssDNA contacts with genomic distance did not follow the genome-average

probability of dsDNA contacts as a function of genomic distance ($P_c(s)$), but instead decayed

more slowly (Figures 2D).

Examination of individual ssDNA sites revealed that DSB-proximal ssDNA contacts were broadly

distributed in *cis*, only enriched 10-fold at the highest over the genome average (Figure S3D). In

contrast, DSB-distal ssDNA sites exhibited a strong contact enrichment with DSB-flanking chromatin,

which decayed following the genome-averaged $P_c(s)$ (Figure 2E and S3D). More specifically, the

proximity to the resection front correlated with frequent ssDNA contacts with chromatin in *cis*, as well

as across the resected region (e.g. compare R8073 at 2 and 4h, Figure S3E). These observations

suggest that ssDNA sites are progressively relieved from constraints confining most contacts in *cis*

over time, which correlates with the increasing distance to the resection front.

22 **DSB-proximal and DSB-distal ssDNA regions are differently emancipated from a cis-**

23 **acting spatial anchor**

The Rabl organization of yeast chromosomes is defined by the anchoring of centromeres at the spindle-

pole body (SPB) via microtubules and of telomeres at the nuclear envelope, whose distance from the

SPB scales with chromosome arm length 34-39. This organization spatially constrains surrounding

chromatin motion, promotes clustering between ~100 kb centromeric regions and inhibits centromere-

telomere contacts 37,40-44 (Figure 1F). The DSB site, positioned 35 kb left of CEN5, is subjected to

such constraint (Figure 1A). Accordingly, ssDNA sites preferentially engaged centromeres in *trans*,

especially for CEN5-associated DSB-distal ssDNA sites, yet to a lower extent than undamaged dsDNA

sites (Figure 2A and S3F). Conversely, DSB-proximal ssDNA sites engaged telomeres of long
chromosome arms more frequently than DSB-distal sites (Figure S3G). These contact profiles
depended on centromere clustering at the SPB, as treatment with the microtubule-depolymerizing drug
nocodazole (i) reduced ssDNA contacts with centromeres, (ii) largely relieved the inhibition of
interactions with telomeres of long chromosome arms, and (iii) abolished the distinction between DSB-
proximal and -distal ssDNA sites, especially for CEN5-associated ssDNA sites (Figures S3H-M).
These results show that DSB-proximal ssDNA is less constrained by a spatial anchor relative to a DSB-
distal ssDNA sites.

ssDNA stiffening projects DSB-proximal sites away from the resection front and the
initial chromatin environment

To interpret the observed changes in *cis* and *trans* contacts at ssDNA sites at varying distances from
the DSB, we propose a theoretical framework where we modeled the NPF as a polymer emanating
from the resection front and fishing for contacts (Figure 2F, see Methods and Supplementary Code
1 for details). We assumed that ssDNA is a homogeneous semi-flexible worm-like chain characterized
by its persistence length Lp - which is the typical length scale below which the polymer tends to remain
linear - and its contour length Lc - which is dynamically evolving as resection progresses. Using a
simple Gaussian contact probability model coupled to a stochastic description of resection, our
framework allowed to predict the ssHiC contacts made by resected ssDNA regions, knowing the
distribution of dsDNA contacts in normal conditions. In particular, we computed the expected fraction
of *trans* contacts and the distribution of *cis* contacts as a function of the distance between the ssDNA
site and the DSB (Figure 2F, Supplementary Code 1). More rigid ssDNA leads to an increase in the
*trans* fraction and to a relative increase in long-range *cis* contacts. Given the typical contour length of
an ATP-bound RecA/Rad51 filament (~0.5 nm per nt of ssDNA) 15 and the inferred dynamics of the
resection front (speed ~3.9 kb/h) 45,46, the value Lp ≈ 20 nm yielded *trans* contacts and *cis* contact
profiles in good quantitative agreement with that observed 4h post-DSB induction (Figure 2G). This
apparent Lp is at least an order of magnitude greater than that of protein-free or RPA-coated ssDNA in
vitro 47-50. It indicates that ssDNA is stiffened in the context of DSB repair by HR in cells, leading to
the distinct contact profiles of DSB-proximal and DSB-distal ssDNA sites (Figure 2H).

ssDNA stiffening by Rad51 promotes genome-wide homology search

We evaluated the contribution of Rad51 in regulating ssDNA stiffness and genome-wide ssDNA
contacts at 2 and 4h post-DSB induction in a *rad51Δ* mutant. Absence of Rad51 did not markedly
affect resection, DSB end-tethering, metaphase-arrest, cohesin-mediated loop folding, chromosome
individualization, or the Rabl chromosome organization 25 (Figure 3A and S4A-E).
The two DNA binding sites of RecA/Rad51 bridge the ssDNA guide and a dsDNA molecule within synaptic complexes, unlike RPA. Once crosslinked, such Rad51-mediated ssDNA-dsDNA association will lead to the ligation events reported by Hi-C<sup>51</sup>. Consistently, the amount of captured ssDNA fragments decreased 2- to 3-fold in a *rad51Δ* mutant compared to WT cells (**Figure 3A**). Calibration of the contact distribution on the capture efficiency of each ssDNA fragment enabled determining the absolute gain and loss of contacts relative to the WT context (**Methods**). *Trans* contacts decreased ~3-fold in the absence of Rad51 for DSB-proximal ssDNA sites, back to levels observed for dsDNA sites (**Figure 3B, C** and **S4F**). Long-range *cis* contacts decreased or remained constant on both sides and across the DSB region (**Figure S4G**). Contacts in *trans* decreased everywhere except at the spatially-associated centromeres (**Figure 3B** and **S4F, I-J**). Contacts with the donor site were abolished (**Figure 3B** and **S4F**). Consequently, the engagement of distant chromosomal regions by ssDNA ends are not due to the rupture of the dsDNA polymer, but instead reflects ongoing homology search by the Rad51-ssDNA NPF.

Unlike in WT cells, the fraction of *trans* contacts as well as the distribution of contacts in *cis* remained similar for all ssDNA sites (**Figure S4H**) and followed the genome-averaged $P_c(s)$, irrespective of their distance to the DSB end in *rad51Δ* cells (**Figure 3D**). Distant ssDNA sites are thus spatially associated and at a null distance from the resection front in the absence of Rad51. These observations show that Rad51 stiffens ssDNA, which otherwise collapses upon RPA binding in *rad51Δ* cells (**Figure 3E**). This stiff Rad51-ssDNA structure projects DSB-proximal sites away from the DSB-borne chromatin environment and promotes genome-wide homology search (**Figure 3E**).

## Exo1-dependent generation of long ssDNA promotes genome-wide homology search

Since long-range resection produces extensive ssDNA for NPF assembly, we addressed its contribution to genome-wide homology search by determining the distribution of ssDNA contacts in *exo1* mutants¹.

The *exo1Δ* and a catalytic-deficient *exo1-D173A* (hereafter *exo1-cd*) mutants gave highly similar results and were pooled (**Figure S5A-G** and see below). The rate of long-range resection was reduced from ~3.9 kb/h in WT cells to ~1.5 kb/h in Exo1-deficient cells (**Figure 4B, Supplementary Code 1**).

As previously noted, Exo1 deficiency also caused a loss of DSB end-tethering, which resulted in the detachment of the centromere-devoid chromosomal fragment<sup>25,52</sup> (**Figure 4A and S5A-G**). However, the Rabl organization, metaphase-arrest, cohesin-mediated loop organization and chromosome individualization were not affected in Exo1-deficient cells<sup>25</sup> (**Figure 4A and S5A-E**).

As expected, Exo1 deficiency led to a loss of ssDNA contacts at DSB-distal regions as they remained non-resected (**Figure 4B**). In contrast, resected DSB-proximal ssDNA sites yielded contacts
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enrichment equivalent to WT (Figure 4B), indicating no notable defect in dsDNA engagement by the NPF in *exo1* mutants. The distribution of ssDNA contacts was strongly altered in Exo1-deficient cells. First, the left NPF contacted the genome in a uniform manner, owing to the loss of end-tethering in these mutants, while contacts between the right NPF and centromeres were exacerbated compared to WT cells (Figure 4C and S5G-H). Second, NPFs were defective in engaging dsDNA in *trans* (Figure 4D). Third, among this reduced fraction of *trans* contacts, interaction with long-arm subtelomeres by the centromere-bound NPF were depleted, similar to undamaged dsDNA sites (Figure S5I). These observations indicate a defect for the NPF in contacting distant dsDNA sites.

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Conversely to this decrease of *trans* contacts, ssDNA contacts by both DSB ends increased with chromatin in *cis* in a side-specific manner (Figure 4E and S5J). The *cis* contact profiles followed the genome-averaged $P_c(s)$ more closely than in WT cells (Figure 4F). They also differed from that observed in a *rad51Δ* mutant, in which all sites exhibited similar slopes to the genome-averaged $P_c(s)$ (Figure 3E). The Gaussian contact probability model satisfyingly accounted for both the fraction of *trans* contacts and the distribution of *cis* contacts with $L_p \approx 17$ nm (Figure 4G-H), very similar to the persistence length obtained in WT cells ($L_p \approx 20$ nm, Figure 2H-I). Consequently, NPF properties are not altered in Exo1-deficient cells.

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Finally, the slope of *trans* contacts as a function of the distance to the right resection front was similar for Exo1-deficient and WT cells (Figure S5K; detachment of the left chromosome fragment prevented a straightforward comparison of the contacts made by the left DSB end). The increased propensity to access genomic regions in *trans* thus reflects the genomic distance between a ssDNA site and the resection front, not the time spent as ssDNA. Altogether, these observations show that long-range resection mediated by Exo1 progressively drives genome-wide homology search by providing increasingly long ssDNA substrates for assembly of Rad51-ssDNA NPF (Figure 4I).

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## Cohesin-mediated chromatin looping focuses search by nascent Rad51-ssDNA NPFs within flanking chromatin loops

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We previously showed that cohesin-mediated loop folding inhibits homology search in *trans* using DSB-flanking dsDNA Hi-C contacts and quantification of D-loop formation of a DSB-proximal sequence at *cis* and *trans* ectopic donors <sup>25</sup>. This inhibition was more pronounced at 2h than at 4h post-DSB induction, which suggested a decreasing influence of cohesin-mediated loop folding on homology search by DSB-proximal sites over time <sup>25</sup>. We addressed the role of cohesin in restricting genome-wide ssDNA contacts upon auxin-induced depletion of the Scc1 subunit of cohesin <sup>25</sup> (Figure 5A). As shown previously, Scc1 depletion abolishes sister chromatid cohesion, chromatin loop folding and
chromosome compaction while retaining the checkpoint-mediated G2/M-arrest<sup>25,53,54</sup> (**Figure S6A-D**). Partial Scc1 depletion relieved an inhibition on *trans* contacts by all ssDNA sites, without markedly affecting the overall ability of the NPF to engage dsDNA (**Figure 5B and S6E-G**). No such relief was observed in a *rad51Δ* mutant background, which again exhibited a defect in contacting dsDNA (**Figure 5B and S6G**). Consequently, cohesin inhibits NPF interactions in *trans*. This cohesin-mediated inhibition was particularly pronounced for DSB-distal NPF sites, while DSB-proximal sites exhibited comparable levels of *trans* contacts at both 2 and 4h post-DSB induction (**Figure 5B**). Scc1 depletion did not affect the distribution of *cis* contacts by DSB-proximal sites, which extended at much longer range than the genome-wide P<sub>c</sub>(s) in a Rad51-dependent manner, as in WT cells (**Figure 5C and S6H**). Similarly, DSB-distal sites behaved as in WT cells and followed the genome-averaged P<sub>c</sub>(s) upon Scc1 depletion (**Figure 5C and S6H**). Consequently, cohesin inhibits long-range *cis* and *trans* homology search by DSB-distal sites within ~10 kb from the resection front.

Examination of individual ssDNA sites further revealed that the NPF region closest to the resection front contacted nearby chromatin predominantly at cohesin-enriched sites (L9924 and L17213 at 2 and 4h post-DSB induction, respectively; **Figure 5D and S6I**). NPF sites further away from the resection front progressively contacted the chromatin located between cohesin peaks, *i.e.* the inner part of chromatin loops (L6065 at 2h and L15683 and L9924 at 4h; **Figure 5D and S6I**). This observation was also true for DSB-distal sites on the right DSB end, although limited to a single loop flanking the centromere of chr. V (**Figure S6J**), possibly owing to the inability for cohesin to cross the centromere<sup>54,55</sup>. Finally, DSB-proximal sites exhibited little cohesin-dependent *cis* contact enrichment (**Figure 5D and S6K**). Cohesin also promoted interactions across the DSB region (**Figure S6I**). The contact profile appeared broad without clear relation with the position of cohesin-enriched sites, even for ssDNA sites close to the resection front, unlike side-specific contacts (**Figure S6J**). These observations show that cohesin directs homology search in *cis* by DSB-distal ssDNA sites in a side-specific manner.

Whether the role of cohesin involved sister chromatid cohesion was addressed upon depletion of the replication factor Cdc45 prior to releasing cells from their G1 arrest<sup>56</sup> (**Figure 5A**). Cdc45-depleted cells reach G2/M with unreplicated chromosomes but retain chromatin loop folding similar to that observed in WT cells<sup>25,54</sup>. The same cell pellets as those used and controlled for in ref. 25 were reprocessed for ssHi-C. Cdc45 depletion did not relieve the cohesin-mediated inhibition of NPF *trans* contacts, nor did it affect contacts with chromatin in *cis* (**Figure 5E-G**). Consequently, the mechanism by which cohesin regulates DSB-distal NPF contacts is independent of its role in sister chromatid cohesion.
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These observations corroborate our prior conclusions that cohesin-mediated loop folding both (i) inhibits homology search in *trans* upon chromosome individualization, and (ii) promotes homology sampling in chromatin loops extruded near the resection front in *cis*²⁵ (**Figure 5H**). It further shows that this influence is exerted primarily over a ~10 kb-long NPF regions flanking the resection front. Consequently, DSB-proximal sites overcome the cohesin-mediated constraint to genome-wide homology search upon long-range resection progression and NPF growth.

## The Recombination Enhancer (RE) focuses homology search on chr. III

Intriguingly, chr. III was disproportionately engaged by ssDNA sites on both sides of, and at all distances from the DSB (**Figure S7A**). This enrichment was most notable 2h post-DSB induction, indicative of an early HR event (**Figure S7A**). Other small chromosomes (I and VI) were significantly less contacted than chr. III and contact enrichment with chr. III was retained upon nocodazole treatment, ruling out the outsize influence of centromeres clustering on short chromosomes as the sole determinant for this enrichment (**Figure S7B**). Contacts by both ssDNA ends were mainly focused in a ~30 kb region surrounding the Recombination Enhancer (RE), enriched by at least an order of magnitude above average inter-chromosomal contacts (**Figure 6A**). The RE is a discrete genetic element that stimulates HR repair with donor sequences located in its vicinity, inside and outside of the context of mating-type switching⁵⁷⁻⁵⁹. Contact enrichment was also notable in a broader ~100 kb region on the right end side of chr. III (**Figure 6A**), presumably owing to the RE-dependent horseshoe folding of chr. III⁴²,⁶⁰,⁶¹ (and see below **Figure 6C**). Rad51 is required for this ssDNA-RE interaction, indicating that these contacts involve the NPF (**Figure 6B and S7C**). The NPF-RE interaction did not require cohesin (**Figure S7D**). However, it depended on the catalytic activity of Exo1 (**Figure S7E, F**), but whether it pertains to its role in forming extended NPFs, in end-tethering, or in regulating the DNA damage checkpoint⁶²,⁶³ is unclear (see **Discussion** below). Deletion of the RE abolished contact enrichment on chr. III (**Figure 6C**). Conversely, addition of the minimal 700 bp-long RE⁶⁴ in the middle of the 1 Mb-long right arm of chr. IV promoted the engagement by the NPF of this normally infrequently contacted ~30 kb surrounding region at 2 and/or 4h post-DSB induction (**Figure 6D**). Consequently, the RE is required and sufficient for NPF recruitment.

DSB-proximal and DSB-distal NPF segments equally contacted the RE (**Figure 6E**), unlike the DSB-flanking dsDNA regions (**Figure 6F**). It suggests that (i) all regions along the Rad51-ssDNA NPF are equally prone to bind to the RE, and (ii) that this NPF-RE binding takes place in an extended NPF (**Figure 6G**).
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These results reveal the existence of a mechanism for local homology search enhancement upon direct interaction between a specialized genetic element and the Rad51-ssDNA NPF that can operate in *trans*.

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**Homology search is partly coordinated between NPFs on both DSB ends**

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The contact profiles of the left and right DSB ends are not super-imposable, yet they differ less than upon loss of DSB end-tethering in Exo1-deficient cells, suggesting that Rad51-ssDNA segments are associated a substantial fraction of the time, or in a substantial fraction of the cell population, during homology search ²⁰ (**Figure 2A, 4C, and 4E**). In order to address this possibility, we focused on contacts with the donor site, which has homology with the left DSB end only. Both left and right NPFs interacted with a ~60 kb region surrounding the donor site, in a homology- and Rad51-dependent manner (**Figures 7A-C, and S8A**). The homology-containing NPF to the left of the DSB contacted the donor region on average 2- to 3-fold more frequently than the heterologous right NPF (**Figure 7A**). DSB-proximal NPF regions exhibited even smaller differences, only ~1.5-fold higher for the left than the right NPF (**Figure 7B**), suggesting that NPF are associated in a co-oriented (i.e. 5'-3') configuration. Loss of DSB end-tethering in Exo1-deficient cells caused a loss of contacts specifically between the heterologous right NPF and the donor (**Figure 4C, 7C, and S8A**). Consequently, left-right NPF interactions are not reestablished once homology has been found by one end. It instead indicates that the majority of the NPFs are associated and carry out homology search in a coordinated manner.

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**Homology identification leads to NPF disruption and loss of DSB end-tethering**

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We previously showed that conventional Hi-C could detect homology- and Rad51-dependent interactions between the DSB-flanking dsDNA regions and the donor ²⁵ (**Figure 2A, 7D, and S8B**). Unlike ssDNA contacts, they were not detected 2h post-DSB induction, and did not involve the right DSB end (**Figure 7D**). Furthermore, DSB-distal ssDNA sites interacted with the donor region as frequently as DSB-proximal sites, specifically on the homology-containing end (**Figure 7B**). These observations indicate that homology recognition leads to a collapse of the homology-containing NPF over time, bringing in proximity of the donor site the DSB-distal ssDNA sites and the dsDNA region flanking the resection front (**Figure 7H**). The absence of contacts between the donor region and DSB-distal ssDNA and dsDNA sites on the heterologous end (**Figure 7B, D, H**) suggested a loss of end-tethering occurring concurrently with the homology-containing NPF collapse. Accordingly, a partial loss of end-tethering occurred between 2 and 4h post-DSB induction in a donor-dependent (**Figure 7E**) and Rad51-dependent manner (**Figure S4A and S5A**).

31

32

**NPF collapse upon homology identification occurs prior to D-loop extension by PCNA-Polδ**
The kinetics of D-loop formation (2 to 4h) and extension (2 to 6h) in this strain³² and the biased DSB-flanking dsDNA and DSB-distal ssDNA contacts in the direction of D-loop extension suggested that NPF collapse could occur prior to, or upon the initiation of displacement DNA synthesis by PCNA-Polδ (Figure S8C). To distinguish between these possibilities, we depleted AID-tagged Pol3 alone or in combination with the PCNA clamp loader subunit Rfc1 after S-phase completion (Figure S8D). We previously showed in these strains that this single and double depletion strategy led to a 3- and 10-fold decrease of D-loop extension, respectively¹³. Both depletion conditions yielded highly similar results. Pol3±Rfc1 depletion did not affect G2/M-arrest, DSB resection, chromatin folding, and chromosome individualization (Figure S8E-I). The contact profiles of both the left ssDNA sites and the flanking dsDNA region were not affected by depletion of Pol3±Rfc1 (Figure 7F and S8J). Consequently, the homology-containing NPF collapse occurs independently of PCNA-Polδ recruitment at the D-loop, and thus of D-loop extension.

The asymmetric ssDNA and dsDNA contact profile in the direction of D-loop extension observed both for dsDNA and ssDNA sites 4h post-DSB induction (Figure S8C) was abolished upon Pol3±Rfc1 depletion (Figure 7G and S8J). It indicates that a fraction of the donor contacts originates from extended D-loops, and that D-loop extension occurs in the context of a disrupted filament (Figure 7H).

Finally, Pol3±Rfc1 depletion did not restore the contacts between the right dsDNA end and the donor (Figure S8K). Consequently, the donor-dependent loss of end-tethering results from a homology-dependent event preceding the initiation of D-loop extension, possibly upon or following DNA strand invasion (Figure 7H). Surprisingly, Pol3±Rfc1 depletion exacerbated the loss of DSB end-tethering observed in WT cells (Figure S8K). It suggests the existence of a PCNA-Polδ-dependent mechanism that holds the two ends together at the donor site, at least in our system in which homology is provided to a single end only.

## Discussion

### Regulated expansion of homology search and its determinants

Homology search is not a uniform process along the genome, the NPF, and over time. Initially local, it progressively expands to become genome-wide, a process orchestrated by independent activities imparted by chromatin folding, chromosome organization, DSB end-tethering, specialized genetic elements, long-range resection, and the extended structure of the Rad51-ssDNA NPF itself (Figure 7I).
Nascent, short Rad51-ssDNA NPFs present close to the resection front sample the proximal chromatin environment. This proximal sampling is focused at chromatin loop bases by cohesin, which accumulates in the DSB region in both yeast and human cells <sup>25,65-70</sup>. Such involvement of cohesin in the initial steps of homology search likely reflects a primary attempt to achieve inter-sister repair, the preferred template in somatic cells <sup>71</sup>. The prevailing assumption that inter-sister repair rapidly occurs between two promiscuously held molecules has been challenged by recent cytological and genomic evidence, which revealed that (i) points of cohesion between sister chromatids are infrequent and staggered in both yeast and human cells <sup>72,73</sup> and (ii) sister loci are ~500 nm distant on average in G2 human cells <sup>74</sup>. Although our system does not allow to study sister chromatid interactions and repair, we suspect that targeting of nascent NPFs at loop bases provides a mechanism to actively direct the search on the sister chromatid. Besides locating the NPF at specific sites that also correspond to inter-sister contact points (i.e. at TAD borders or loop bases <sup>72,73,75</sup>), such mechanism is likely to also require loop extrusion on the sister chromatid to bring potential donor molecules in proximity to the NPF. Cohesin may thus turn homology search from an inefficient tridimensional diffusive process into a pseudo-1D walk on the sister chromatid. Such walk is only possible in the context of short or highly segmented NPFs that remain in the immediate proximity of the inter-sister association point from which loop extrusion proceeds.

Long-range resection gradually provides ssDNA substrates for assembly of longer NPFs. By virtue of their rigidity <sup>16,76</sup>, these NPFs progressively project DSB-proximal search units away from the initial chromatin environment, eroding the influence of cohesin on the search process despite the maintenance of cohesin-mediated loop folding <sup>25</sup>. Hence, DSB-proximal and DSB-distal regions of the NPF simultaneously sample distinct genomic regions and nuclear area. This genomics-based evidence supported by modelling parallels the extended structures stretching across the nucleus recently observed in *S. cerevisiae* with a functional GFP-tagged version of Rad51 <sup>20</sup>.

Finally, homology search is conducted by (at least partly) paired, co-oriented NPFs, as revealed by interactions of both ssDNA ends with a region of homology to only one DSB end. This paired NPF configuration is consistent with end-tethering determined with Hi-C and cytologically with fluorescently-tagged Rad51 and other HR proteins, as well as fluorescent operator-repressor arrays in the flanking dsDNA region <sup>25,26,29,30,52,77</sup>. Homology identification leads to NPF collapse specifically at the homology-containing end. This collapse is likely to limit coincidental homology identification at multiple genomic sites along a single NPF, and thus precludes the cascade of rearrangements initiated by multi-invasion DNA joint molecules <sup>10,13</sup>.

**The possibility of genome-wide homology search lies in NPF structure**
1 Chromatin loci exhibit a sub-diffusive behavior limiting their ability to explore the nuclear space ⁴⁰.
2 The increased confinement radius observed upon break induction at both damaged and undamaged
3 sites suggested that the possibility of nucleus- and genome-wide homology search resided in changes
4 to chromatin property ²⁹,³⁰. This paradigm was recently challenged with the identification of a DSB
5 mobility-defective *uls1Δ* mutant that achieved inter-chromosomal ectopic DSB repair with wild-type
6 kinetics ⁷⁸. Differently, the present work and recent cytological observations of functional
7 fluorescently-tagged RecA/Rad51 proteins in bacterial and *S. cerevisiae* cells ¹⁸,²⁰,²¹ indicate that the
8 possibility of genome-wide homology search lies in their conserved, rigid structures well-characterized
9 *in vitro* ². Consequently, the length of a Rad51-ssDNA NPF not only multiplies search units but also
10 expands the searchable genome fraction. Specifically, projection of NPF segments across the nuclear
11 space entails the presence of at least one long Rad51 structure between the DSB end and the resection
12 front. Since the segment length distribution depends on the nucleation, growth and dissociation rates
13 of RecA/Rad51 on ssDNA ⁷⁹, factors such as mediators and regulators of NPF stability as well as
14 species-specific differences in RecA/Rad51 nucleation/elongation rates likely dictate whether and with
15 which kinetics the transition from local to genome-wide search occurs ⁸⁰⁻⁸².

16 Our Gaussian polymer model consistently accounts for the observed proportion of *trans* contacts and
17 the distribution of *cis* contacts with an apparent ssDNA persistence length of ~20 nm. It is at least an
18 order of magnitude higher than that of protein-free or RPA-coated ssDNA but an order of magnitude
19 lower than that of ATP-bound RecA/Rad51-ssDNA NPF measured *in vitro* ¹⁶,¹⁷,⁴⁹,⁵⁰,⁷⁹,⁸²,⁸³. This average
20 value likely reflects a heterogeneous ensemble of ssDNA states in cells, with collapsed RPA-bound
21 segments, contracted Rad51-ADP bound segments, and extended Rad51-ATP-bound segments co-
22 existing on the same molecule and in varying proportion in the cell population. Consistently, Rad51-
23 GFP structures dynamically alternate between globular and extended conformations in cells ²⁰.
24 Correcting for the fraction of cells lacking an elongated Rad51-GFP structure, our model predicts a
25 NPF contour length of ~1.1 µm (**Supplementary Code 1**), closely matching sizes determined
26 cytologically ²⁰.

27 Long-range resection promotes genome-wide homology search

28 The amount of ssDNA generated by long-range resection largely exceeds the length of homology
29 needed for efficient ectopic repair by HR ⁸⁴⁻⁸⁶, which questioned its biological relevance ⁶². Here, we
30 show that Exo1 promotes genome-wide homology search by DSB-proximal ssDNA. It likely
31 represents and adaptation of the search strategy in the event of a prolonged failure to repair using the
32 sister chromatid ⁸⁷. Consistently, long-range resection is required only when the donor is provided >50
1

kb away from the DSB site or on another chromosome⁶², *i.e.* outside the typical range of high ssDNA contacts determined here in WT cells early post-DSB induction and in *exo1* mutants. This expansion mechanism driven by long range resection is likely conserved, as resection tracts at replication-dependent breaks also extend for tens of kilobases in repair-defective human cells⁸⁸.

2

3

4

5

Since as little as 2 kb of ssDNA is sufficient to assemble a 1 µm-long NPF (*i.e.* the diameter of a yeast nucleus), ssDNA is unlikely to be limiting for extended NPF assembly, even in the absence of Exo1. Consequently, and as discussed above, we favor the view that most NPF segments are short, and that the excess of ssDNA produced by long-range resection increases the probability of assembling at least one long (*i.e.* >1 kb ~ 500 nm) segment (**Figure 7I**). Hence, the assembly of long, continuous Rad51-ssDNA segments is likely a rate-limiting step for genome-wide homology search in *S. cerevisiae*.

6

7

8

9

10

11

## Role of DSB end-tethering in coordinating homology search by the two NPFs

12

Maintenance of end-tethering involves several handovers during the course of DSB repair: initially mediated by the Mre1-Rad50-Xrs2NBS1 (MRXN) complex prior to and upon short-range resection, it is followed by Exo1 and the Ddc1-Mec3-Rad17 checkpoint clamp (human 9-1-1 clamp) independently of its checkpoint function ²⁵,²⁷,⁵². This tethering at the level of the resection front is required for coordinated search by both NPFs. Such coordination is supported by the predominance of single Rad51-GFP structures over branched and complex structures observed cytologically²⁰.

13

14

15

16

17

18

Functionally, pre-association between DSB ends facilitates SDSA repair steps downstream of D-loop extension ⁸⁹,⁹⁰. Here we show that end-tethering promotes coordinated homology search between the two ends. Such coordination likely promotes concurrent donor identification and/or invasion by both DNA ends, a possible precursor of double Holliday junction formation ⁹¹.

19

20

21

22

Intriguingly, in the absence of homology to the second end, end-tethering is progressively lost, coincidentally with a disruption or collapse of the homology-containing Rad51-ssDNA NPF. This observation extends the sequence of handovers required for the maintenance of DSB end-tethering taking place along the HR pathway at the synaptic step upon homology identification of the same donor site by both DSB ends. Absence of shared homology is likely to signal an ectopic invasion event, and loss of end-tethering in this context may inhibit the formation of repeat-mediated rearrangements. PCNA-Polδ also promoted end-tethering in our system lacking homology to the second end. The biological relevance and mechanism of this association remain to be determined.

23

24

25

26

27

28

29

30

## Not all genomic sites are equal for homology search
We show that the genome is not equally sampled during homology search, and that multiple mechanisms exist to enhance it at specific genomic loci, conditionally or constitutively: cohesin promotes sampling of regions at the base of chromatin loops (and presumably the sister chromatid) by ssDNA sites newly generated by resection; centromere clustering promotes search on other centromeres; and DSB end-tethering enhances homology search around the donor site if homology has been identified by one of the two end. We identified a fourth mechanism specific of chromosome III that engages pioneer, extended NPFs. It relies on the RE, an autonomous genetic element involved in biasing donor usage in mating-type switching or ectopic repair in *MATA* cells <sup>57,59,64</sup>. Our observations strongly suggest that the RE stimulates recombination with nearby donor by engaging the NPF, which locally enhances homology search.

The RE function depends on its binding by multiple copies of the conserved fork-head-family transcription factor Fkh1 <sup>59,92,93</sup>, otherwise present as a monomer at gene promoters throughout the genome <sup>94,95</sup>. It raises the possibility that Fkh1 plays a general role in homology search outside the RE, by regularly seeding homology sampling events in promoters. Such mechanism may drastically reduce search complexity and could promote HR fidelity by occluding repeated elements.

## Study of other DNA metabolic processes with ssHi-C

ssDNA is a hallmark of various fundamental DNA metabolic processes. By directly targeting ssDNA, ssHi-C uniquely enables the site-specific determination of the contacts made by DNA while it is being involved in such processes, even if only a subpopulation of cells exhibits ssDNA at the site of interest. Since ssHi-C operates according to Watson-Crick base-pairing rules and is protein-independent, it can be applied in any organism for which the genome sequence is known, without a need for specific reagents (e.g. antibody) or genetic engineering. Consequently, ssHi-C is a universal method for the study of the spatial aspects of DNA metabolic processes involving ssDNA.

## Limitations of the study

As discussed previously, our experimental system does not allow to study sister-based repair as (i) both sister chromatids are cut and (ii) inter-sister interactions cannot be resolved by our Hi-C and ssHi-C protocols. The ssHi-C methodology is also limited in that it requires oligonucleotides specific to the ssDNA of interest. As such, it is blind to other ssDNA regions than that of interest. Consequently, it is ill-suited to study the repair of random breaks or of other processes in which the location and polarity of ssDNA is unknown. Finally, as noted in the results section, not all ssDNA sites are equally well restored. It suggests that as-of-yet unidentified aspects of the annealing oligonucleotide design and/or steps of the protocol can be improved to maximize and uniformize recovery of ssDNA contacts.
1 Acknowledgements

2 We thank Piazza, Jost and Koszul lab members for stimulating discussions, Wolf-Dietrich Heyer and
3 Angela Taddei for their critical reading of the manuscript, and Romain Koszul for critical reading of
4 the manuscript and for sharing expertise and protocols. We are grateful to Karine Dubrana for sharing
5 unpublished data regarding intra-chromosomal distances in G2/M cells.

6 This research was supported by the CNRS as part of its Momentum program and the European
7 Research Council (ERC) under the European Union's Horizon 2020 to A.P. (ERC grant agreement
8 851006) and the Agence Nationale de la Recherche to DJ (ANR-21-CE13-0037-02, ANR-22-CE12-
9 0035-03).

10 Author contributions

11 Conceptualization: AP; Experiments: AD, JS, AT, PM, AP; Data analysis: NM, AP; Software
development: NM, LA, LM; Modeling: DJ; Data interpretation: AP, DJ; Supervision: AP, LM;
13 Funding Acquisition: AP; Writing: AP.

14 Declaration of interest

15 A patent "A METHOD FOR IDENTIFYING GENOMIC INTERACTIONS" that covers aspects of
16 the ssHi-C methodology has been submitted on September 11, 2023 by the CENTRE NATIONAL DE
17 LA RECHERCHE SCIENTIFIQUE under PCT application number PCT/FR2023/051384, with AD
18 and AP as listed inventors.
1 Main figure titles and legends

2 **Figure 1: Genome-wide mapping of ssDNA and dsDNA contacts with ssHi-C.**

3 (A) Experimental system in haploid *S. cerevisiae*.

4 (B) Cell synchronization, DSB induction and cell fixation protocol.

5 (C) Hi-C coverage following DSB induction. Long-range resection proceeded at 3.9 kb/h (see Supplementary Code 1). Data is mean±SEM of 4 biological replicates.

7 (D) Rationale and steps of ssHi-C.

8 (E) Annealing oligonucleotides used in this study. The black probes do not have corresponding capture oligonucleotides, and correspond to negative controls in (F).

10 (F) Hi-C and ssHi-C contact maps of the whole genome 4h post-DSB induction in WT cells (APY266). Insets: Inter-chromosomal contacts between chr. V (black arrow) and chr. II to IV. Absolute and relative coverage vs. undamaged cells show contact enrichment at dsDNA and ssDNA sites in ssHi-C. Maps are merge of 3 biological replicates subsampled at 19 million contacts. Bin: 20 kb.

14 (G) ssHi-C retrieves yeast chromosome organization features in addition to ssDNA-specific contacts.

16 **Figure 2: DSB-proximal ssDNA sites emancipate from *cis* spatial constraints over time.**

17 (A) Average genome-wide contact distribution of the left and right DSB ends determined with Hi-C (left; dsDNA only, viewpoint 5 kb away from the DSB site) and ssHi-C (right; ssDNA only, average of all ssDNA sites) 4h post-DSB induction in WT cells (APY266). A control dsDNA site on chr. VIII, located at the same distance to a centromere as the DSB (Figure S1C), is shown for comparison. Dotted lines: centromeres. Anchors: viewpoints. Orange arrow: donor. Data represent the average of 4 biological replicates smoothed over 30 kb. mt: mitochondria. 2µ: 2-micron plasmid.

23 (B) *Trans* contacts for dsDNA and ssDNA sites determined in the DSB region (right) and at non-damaged sites genome-wide (left) with Hi-C and ssHi-C in WT cells. Data points: individual ssDNA sites (ssHi-C) or 1 kb dsDNA bins (Hi-C).

26 (C) Top: Hi-C coverage. Bottom: Proportion of ssDNA contacts in *trans* as a function of the distance to the DSB. Dotted lines: resection fronts.

28 (D) LOWESS regression of average contacts made by ssDNA sites on the left DSB end in *cis* compared to the genome-averaged $P_c(s)$ at 2 and 4h post-DSB induction. Dotted line: Genome-wide dsDNA $P_c(s)$.

30 (E) LOWESS regression of individual contacts made by ssDNA sites on the left DSB end in *cis* at 4h post-DSB induction in WT cells. Other legends as in (D).
1

2

3

4

5

6

7

8

(F) Left: "Fishing line" modeling rationale. The red polymer represents ssDNA emanating from the dsDNA polymer. Lp: persistence length. Right: Predicted fraction of *trans* contacts at increasing distance from the resection front with different Lp.

(G) Predicted distribution of *cis* contacts with Lp = 20 nm.

(H) Differential DSB-distal and DSB-proximal contacts explained by ssDNA stiffening.

**Figure 3: Rad51 promotes genome-wide homology search by DSB-proximal ssDNA sites by stiffening ssDNA.**

9

10

(A) Hi-C coverage (top) and amount of dsDNA (left) and ssDNA (right) contacts retrieved in a *rad51Δ* mutant (APY679) relative to WT cells (bottom). Data: mean ± SEM of 2 biological replicates.

11

12

(B) Average genome-wide contact distribution of the left and right ssDNA sites 4h post-DSB induction in WT cells and *rad51Δ* cells (APY679). Other legends as in **Figure 2A**.

13

14

(C) *Trans* contact frequency relative to WT cells for individual ssDNA sites (right) and 7 control dsDNA sites (left) in a *rad51Δ* mutant. Data: mean ± SEM.

15

16

(D) LOWESS regression of the average (left) and individual (right) *cis* contacts made by ssDNA sites on the left DSB end, compared to the genome-wide *Pc(s)* in WT and *rad51Δ* cells.

17

18

(E) Model: Rad51 stiffens ssDNA, which promotes genome-wide homology search by DSB-proximal ssDNA sites.

19

**Figure 4: Exo1 catalytic activity promotes genome-wide homology search.**

20

21

22

(A) Left: Hi-C contact maps of chr. V in WT and *exo1-D173A* (APY536) cells 4h post-DSB induction. Right: Ratio map.

23

24

25

(B) Hi-C coverage (top) and amount of dsDNA (left) and ssDNA (right) contacts retrieved in *exo1Δ* and *exo1-D173A* mutants (APY1160 and APY536, respectively) relative to WT cells (bottom). Data: mean ± SEM from a pooling of both mutants (one biological replicate each).

26

27

(C) Average genome-wide contact distribution of the DSB-proximal left and right DSB ends 4h post-DSB induction in WT cells and pooled Exo1-deficient cells. Other legends as in **Figure 2A**.

28

29

(D) Average *trans* contact frequency for individual ssDNA sites (right) and 7 control dsDNA sites (left) at 2 and 4h post-DSB induction in WT and Exo1-deficient cells in (C). Data: mean ± SEM.

30

(E) Zoom of the ssHi-C data in (C), smoothed over 4 kb.

31

32

(F) LOWESS regression of the average (top) and individual (bottom) *cis* contacts made by ssDNA sites on the left DSB end, compared to the genome-averaged *Pc(s)* in WT and Exo1-deficient cells.
1

(G-H) Predicted fraction of *trans* contacts (G) and distribution of *cis* contacts (H) at increasing distance from the DSB site with L~p~ = 17 nm compared to experimental data from (D).

2

3

(I) Model for the Exo1-driven genome-wide expansion of homology search.

4

5

**Figure 5: Cohesin directs homology search by DSB-distal ssDNA sites in cis.**

6

(A) Experimental schemes for depletion of AID-tagged Scc1 and Cdc45.

7

(B) Average *trans* contact frequency for individual ssDNA sites (right) and 7 control dsDNA sites (left) at 2 and 4h post-DSB induction in WT (n=4), Scc1-depleted cells (APY1481) and Scc1-depleted *rad51Δ* cells (APY1500).

8

9

10

(C) LOWESS regression of the average (top) and individual (bottom) *cis* contacts made by DSB-proximal (left panel) and DSB-distal (right panel) ssDNA sites on the left DSB end, compared to the genome-wide dsDNA P~c~(s).

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12

13

(D) Side-specific *cis* contact profiles of individual ssDNA sites in WT and Scc1-depleted cells 4h post-DSB induction. The calibrated Scc1-6HA ChIP-Seq profile obtained in this system 4h post-DSB induction in WT cells are from ref. 25. Dotted lines: position of cohesin peaks. Arrows: cohesin-dependent enrichment of ssDNA contacts.

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17

(E) Average *trans* contact frequency 4h post-DSB induction in untreated WT cells, Cdc45-AID-tagged cells (APY513, +DMSO), or Cdc54-depleted cells (+IAA) (n = 2 each).

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19

(F) LOWESS regression of the average *cis* contacts made by DSB-proximal ssDNA sites on the left DSB end compared to the genome-averaged P~c~(s) in WT, Cdc54-tagged and Cdc45-depleted cells.

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21

(G) Side-specific *cis* contact profiles for the DSB-distal L15683 ssDNA site in WT, Cdc54-tagged and Cdc45-depleted cells 4h post-DSB induction. Other legends as in (D).

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23

(H) Model for the interaction of DSB-distal ssDNA sites with side-specific cohesin-mediated chromatin loop in *cis*.

24

25

**Figure 6: The recombination enhancer stimulates homology search in *trans* on chr. III.**

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27

(A) ssHi-C contact profiles on chr. I and III at 2 and 4h post-DSB induction in WT cells. From data in Fig. 2.

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29

(B) Pondered ssHi-C contact profiles in *rad51Δ* cells 2h post-DSB induction. From data in Fig. 3.

30

(C) ssHi-C contact profiles in a strain deleted for the RE (APY1548) 2h post-DSB induction.

31

(D) ssHi-C contact profiles on chr. III and IV in WT cells and in cells bearing an additional 700 bp RE element inserted at position 845,464 on chr. IV (APY1497) at 2 and 4h post-DSB induction.

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33

(E, F) Contact profiles of individual ssDNA sites (E) and of dsDNA regions 5 kb away from the DSB (F) on the left and right sides 2h post-DSB induction in WT cells.

34
1 (G) Model for the NPF-RE interaction.

2 Signal smoothed over 4 kb, except in (D), smoothed over 10 kb.

3

4 **Figure 7: Coordinated homology search by both DSB ends and side-specific NPF collapse**
5 **in a donor-dependent manner.**

6 (A) ssHi-C contact profiles in WT cells. From data in **Figure 2**. Data smoothed over 10 kb. Other
7 legends as in **Fig. 2A**.

8 (B) Contact frequency of individual ssDNA sites with the 60 kb donor-surrounding region in the
9 presence (n = 4) and absence (APY358, n = 1) of homology for the left DSB end at that site. a: slope
10 of the linear regression on both side of the DSB. Data: mean ± SEM.

11 (C) Same as (B) in a *rad51Δ* mutant (left) and an *exo1-D173A* mutant (right) 4h post-DSB induction.

12 (D) Hi-C contact profiles of DSB-flanking dsDNA regions 5 kb away to the left and right end of the
13 DSB site. Other legends as in (A).

14 (E) Hi-C ratio map of a strain bearing a donor (APY266) over a strain lacking a donor (APY358) for
15 the left DSB end at 2 and 4h post-DSB induction.

16 (F) Same as in (B) in WT (n=4), Pol3-depleted (APY1352, n=1) and Pol3- and Rfc1-depleted
17 (APY1350, n=1) cells 4h post-DSB induction.

18 (G) ssDNA contact profiles over a 240 kb region surrounding the donor site in WT, Pol3-depleted and
19 Pol3- and Rfc1-depleted cells 4h post-DSB induction. Data smoothed over 10 kb.

20 (H) Model for initial contacts with the donor by both NPF, homology-containing NPF disruption and
21 loss of end-tethering.

22 (I) Model for homology search in *S. cerevisiae*.

23
1 **STAR Methods**

2 **Resources availability**

3 **Lead contact**

4 Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Aurèle Piazza (aurele.piazza@ens-lyon.fr).

6 **Materials availability**

7 *S. cerevisiae* strains generated in this study are available upon request. Strain genotypes are listed in **Table S1**.

9 **Data and code availability**

10 • The raw Illumina paired-end sequencing data have been deposited to the NCBI Sequence Reads Archive (SRA) under bioproject accession PRJNA1139728 and is publicly available as of the date of publication. HiC data in cooler format binned at a 1 kb resolution and unbinned ssHi-C data have been deposited to NCBI Gene Expression Omnibus (GEO) under accession GSE272969 and is publicly available as of the date of publication. The correspondence between figure panels and Hi-C and ssHi-C libraries is provided in **Table S5**. Original western blot images have been deposited at Mendeley (https://data.mendeley.com/datasets/mc3vvgb8pc/1) and are publicly available as of the date of publication. The DOIs are listed in the key resources table.

19 • All original code and reference genomes used for Hi-C (S288c_DSB) and ssHi-C (S288c_DSB_LY_Capture_artificial) alignment have been deposited to Github (https://github.com/Piazzalab/ssHiC_MolCell_2024) and released on Zenodo (https://doi.org/10.5281/zenodo.13149251) and are publicly available as of the date of publication. The sshicstuff pipeline has been deposited to Github (https://github.com/Piazzalab/sshicstuff) and released on Zenodo (https://doi.org/10.5281/zenodo.13149287) and is publicly available as of the date of publication. The stochastic model of resection and the Gaussian polymer model is available as **Code S1**.

28 • Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
29
# 1 Experimental Model and Study Participant Details

## 2 *Saccharomyces cerevisiae* strains

3 Genotypes of *Saccharomyces cerevisiae* (W303 RAD5+ background) strains are listed in **Table S1** and the key resources table. The inducible *HO* expression construct *pGAL1-HO* at *trp1* (chr. IV), the mutagenesis of the endogenous *HO* cut-site at *MAT* (chr. III), the DSB-inducible construct replacing *URA3* (chr. V) and the donor sequence replacing *LYS2* (chr. II) have been described previously ³¹,³².
4
5
6
7 The DSB-inducible construct contains 327 bp of a fragment of the PhiX phage genome flanked by multiple restriction sites (including *DpnII*) making a unique 453 bp sequence pad for annealing of oligonucleotides in ssHi-C, a 2,086 bp-long sequence corresponding to the first half of the *LYS2* gene (+4 to +2,090), and a 117 bp HO cut-site. It is positioned at position 116,167 (S288c coordinates), replacing *URA3*. A 2,086 bp-long ectopic donor (+4 to +2,090 of the *LYS2* gene) is present at its endogenous locus on chr. II.
8
9
10
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12

13 The construct *his3::pADH1-OsTIR1-9Myc::HIS3* for OsTir1 E3-Ubiquitin ligase expression, as well as the Scc1-(Pk3-)AID, Scc1-6HA and Cdc45-(FlagX5-)AID constructs have been described previously ³²,⁵⁴. The *rad51::kanMX*, *exo1::kanMX*, and *exo1-D173A* mutations were previously described ²⁵. The *RFC1-AID-9Myc::hphMX* construct has been described previously ⁹⁶. The *POL3-iAID* construct has been described previously ⁹⁷,⁹⁸. The constructs for *TIR1* and *TetR* expression integrated at *SSN6* have been described previously ⁹⁸. Seamless deletion of the RE on chr. III (R64-2-1 coordinates chrIII:29107-30801) was achieved upon templated CRISPR/Cas9-induced editing ⁹⁹ (**Table S2**). Insertion of the 700 bp RE at position 845464 on chr. IV was achieved by transformation of a strain containing *URA3* at that site with a PCR product amplified with primers RE-atchrIV-845464-F/R (**Table S2**) and selecting for 5-FOA-resistant colonies. Gene deletions, fusions and point mutations were verified by PCR and/or Sanger sequencing, as well as from bam sequence files generated upon alignment of Hi-C or ssHi-C reads. Protein tagging was verified by Western blotting.
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## 25 Method details

### 26 Culture media

27 Liquid YPD (1% yeast extract, 2% peptone, 2% dextrose) and YEP-lactate (1% yeast extract, 2% peptone, 2% lactate) were prepared according with standard protocols ¹⁰⁰.
1 Induction of DSB formation in cells synchronously released in S-phase

2 Exponentially growing cultures in YEP-lactate (30°C) were synchronized at the G1/S transition by
3 addition of 1 µg/ml alpha-factor (GeneCust) every 30 minutes for 3 hours. Cells were washed 3 times
4 with 50 mL of pre-warmed YEP-lactate and released in S-phase in YEP-lactate supplemented with 2%
5 galactose to induce HO expression. The cell-cycle stages were verified by flow cytometry.

6 Protein depletion

7 Crosslinked pellets originating from time courses performed in ref. ²⁵ were reprocessed for ssHi-C.
8 Protein depletion and flow cytometry analysis were reported in ref. ²⁵. Briefly, Cdc45-(FlagX5-)AID
9 depletion was induced in cells synchronized with alpha-factor by addition of 2 mM 3-Indole Acetic
10 Acid (IAA, i.e. Auxin) solubilized in DMSO, or of the equivalent DMSO concentration (0.35% final)
11 as control, 1 hour before release from the G1 arrest.

12 Scc1-(Pk3-)AID depletion was induced at the time of release in S phase by addition of 2 mM IAA (or
13 the equivalent DMSO concentration), together with DSB induction.

14 Inhibition of *POL3-iAID* expression and depletion of Pol3-iAID independently or together with Rfc1-AID-9Myc was conducted similarly to <sup>13,97</sup>. Transcriptional inhibition was induced upon 50 µg/mL doxycycline and protein depletion induced upon 1.5 mM IAA addition 1 hour after S-phase release and HO induction, to allow for bulk DNA replication to take place. Proteins levels were determined by western blot.

19 Flow cytometry

20 Approximately 10⁷ cells were collected by centrifugation, re-suspended in 70% ethanol and fixed at
21 4°C for at least 24h. Cells were pelleted, resuspended in 1 mL of 50 mM sodium citrate pH 7.0 and
22 sonicated 10 seconds on a Bioruptor. After washing, cells were treated with 200 µg of RNase A
23 (Euromedex, cat.9707-C) at 37°C overnight. Cells were then washed and incubated for 30 minutes
24 with 1 mL of 50 mM sodium citrate pH 7.0 with 16 µg of propidium iodide (Fisher Scientific,
25 11425392). Flow cytometry profiles were obtained on a MACSQuant machine and analyzed using
26 Flowing Software 2.5.1.
1 Hi-C and ssHi-C procedures

2 • Annealing and capture oligonucleotide design

3 Annealing oligonucleotides have been designed according to the following rules:

4 o Be 80 nt-long.

5 o Overlap a DpnII restriction site and a MfeI or SspI restriction site located at less than

6 40 bp from the DpnII site.

7 o Asymmetrically positioned relative to the DpnII site so has to ensure sufficient base-

8 pairing near the DpnII site (> 4 bp) and maximize the length of annealed dsDNA

9 produced on the MfeI/SspI-containing side.

10 o Contain 4-6 single nucleotide substitutions introduced as evenly as possible within the

11 MfeI/SspI-containg side. One of these substitutions must inactivate the MfeI/SspI site.

12 Other substitutions are chosen preferentially to inactivate cryptic DpnII (e.g. AATC

13 instead of GATC) and HinfI (e.g. AANTC instead of GANTC). Substitutions are not

14 introduced within 4 bp of the DpnII site. Finally, they must be as evenly distributed as

15 possible.

16 o Oriented so as to anneal to the ssDNA present in the cell.

17 o Be HPLC- or PAGE-purified.

18 o Have their 5' and 3' ends chemically blocked to prevent artefactual biotinylation and

19 ligation in the absence of annealing to, and digestion of, their DpnII target site.

20 Blockers used are 5-methyl-deoxycytidine at the 5' end and the phosphoramidite C3-

21 spacer at the 3' end.

22 Capture oligonucleotides target the dsDNA part restored by the annealing oligonucleotides, apart from

23 control dsDNA capture oligonucleotides that target single dsDNA fragments without specific

24 constrains. They contain a 5' Biotin. Annealing and capture oligonucleotides were synthesized by

25 Integrated DNA technologies. Annealing and capture oligonucleotides sequences are provided in

26 **Table S3** and **S4**, respectively. For an example of the design of the annealing and capture

27 oligonucleotide for the DpnII site L2599 (corresponding to the restriction fragment 18614), see **Figure**

28 **S1B**.

29 • Cell fixation with formaldehyde and EGS
1

Cell fixation is adapted from Micro-C-XL <sup>101</sup>. Approximately 1.5x10<sup>9</sup> haploid cells were fixed with 3% formaldehyde (Sigma-Aldrich, cat. F8775) for 30 minutes at RT at 120 rpm. Formaldehyde was quenched with 330 mM glycine for 20 minutes at RT at 120 rpm. Cells were washed twice with cold water at 3,000 g for 10 minutes. Pellets were split in two tubes and frozen at -80° C.

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Cell pellets were thawed in ice, resuspended in 10 mL of Zymolyase solution (0.25 mg/mL Zymolyase 100T (Carl Roth, cat. 9329), 1 M D-sorbitol (Sigma-Aldrich, cat. S6021), 50 mM Tris-HCl pH 7.5, 10 mM 2-mercaptoethanol (Sigma-Aldrich, cat. M3148)) and spheroplasted at 210 rpm for 18 minutes at 30° C (cells spheroplasting is checked under the microscope and the incubation time adjusted accordingly). Spheroplasts are pelleted at 3,000 g for 10 minutes at 4° C, washed with 4 mL cold 1X PBS, and resuspended in a fresh EGS crosslinking solution (3 mM ethylene glycol bis(succinimidyl succinate) (Fisher Scientific, cat. 10350924) in 1X PBS), and incubated at 30° C for 40 minutes at 100 rpm. EGS was quenched with 400 mM glycine at RT for 5 minutes at 100 rpm. The crosslinked cellular material was pelleted at 3,000 g for 10 minutes at 4° C and washed twice in cold 1X PBS supplemented with anti-protease (Sigma-Aldrich, cat. 11836170001). Pellets were snap-freezed in liquid nitrogen and stored at -80°C.

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* Hi-C procedure with restriction site restoration in ssDNA

17

The crosslinked pellets were thawed in ice and resuspended in cold H₂O supplemented with anti-protease. Between 2 and 5.10⁷ cells were used for the remainder of the procedure. The ssHi-C procedure uses the Arima Hi-C kit (Arima Genomics, cat. A410079), which employs a dual restriction digestion (DpnII and HinfI) that yields a median fragment length of 108 bp in *S. cerevisiae*. The protocol follows manufacturer's instructions with the following modification: after the lysis step, the pool of HPLC- or PAGE-purified annealing oligonucleotides (stock concentration 100 nM each) is added at a final concentration of 7 nM each. Annealing occur during the subsequent incubation at 62° C for 10 minutes.

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* Sequencing library preparation

26

DNA was fragmented into 300-400 bp fragments using Covaris M220 sonicator. Preparation of the libraries for paired-end sequencing on an Illumina platform was performed using the Thermofisher Collibri ES DNA Library Prep Kit for Illumina Systems with UD indexes (cat. A38606024) following manufacturer's instructions. The library was amplified in triplicate PCR reactions using oligonucleotides corresponding to the Illumina sequence adaptors (5'-

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1 AATGATACGGCGACCACCGAGATCTACAC-3' and 5'-

2 CAAGCAGAAGACGGCATACGAGAT-3') and Phusion DNA polymerase (New England Biolabs, cat. M0531) for 11 cycles. PCR products were purified with AMPure XP beads (Beckman-Coulter, cat. A63881) and resuspended in pure H₂O. The Hi-C library is quantified using the Qubit DNA high sensitivity kit (Thermo Scientific, cat. Q32851) on a Qubit 2 fluorometer (Thermo Scientific, cat. Q32866).

7 • ssHi-C library Capture

8 The ssHi-C library consists in an enrichment of ssDNA contacts from the Hi-C library using a panel
9 of biotinylated “capture” oligonucleotides. 500 ng of PCR products were saturated with 2 µg of
10 unlabeled Cot1 human DNA (Invitrogen, cat. 15279-011) and 0.2 nmol of oligonucleotides
11 corresponding to the universal Illumina adaptors (see above) and dehydrated at 60° C for 1 hour in a
12 Speedvac (Thermo Scientific, cat. DNA130-230). DNA was resuspended in 1X xGen Hybridization
13 buffer and 0.15X of xGen Hybridization Enhancer (Integrated DNA Technologies, cat. 10010352),
14 and the pool of biotinylated capture oligonucleotides (stock 1.8 µm each) at a final concentration of
15 0.6 µM. DNA is denatured at 95° C for 5 minutes, and annealing occurs at 65° C for 4h. Biotin
16 pulldown on streptavidin-coated dynabeads C1 (Invitrogen, cat. 65001) was performed according to
17 manufacturer's instructions, and DNA was recovered in pure H₂O. PCR amplification (10 cycles) and
18 DNA purification on AMPure XP beads was carried out as before. The capture procedure was repeated
19 once and the final library was amplified for only 8 cycles.

20 • Optional: elimination of the dsDNA-borne contacts

21 This step eliminates the chimeric molecules involving the fragments of interest (i.e. targeted by
22 annealing and capture oligonucleotides) that were in a dsDNA form in the cell population at the time
23 of crosslink, upon digestion by restriction enzymes whose site was eliminated in the annealing
24 oligonucleotide (rationale **Figure S1A**) 300 ng of PCR products were digested at 37° C for 1 hour with
25 10 units of MfeI-HF and SspI-HF in 1X rCutsmart buffer (New England Biolabs, cat. R3589, R3132
26 and B7204, respectively) and the enzymes heat-inactivated at 65° C for 20 minutes. DNA was purified
27 with AMPure XP beads, resuspended in pure H₂O, and quantified using the Qubit DNA high sensitivity
28 kit (Thermo Scientific, cat. Q32851) on a Qubit 2 fluorometer (Thermo Scientific, cat. Q32866).

29 • Paired-end illumina sequencing
1

ssHi-C libraries were paired-end sequenced (2x150 bp) on an Illumina Novaseq6000 platform by

2

Novogene UK.

3

## Protein extraction and western blotting

4

Protein extracts for western blot were prepared from $5.10^7$ to $10^8$ cells. Cells were lysed in cold NaOH buffer (1.85 N NaOH, 7.5% v/v beta-Mercaptoethanol) for 10 min in ice. Addition of trichloroacetic acid (15% final) for 10 min in ice allowed protein precipitation. After centrifugation at 15,000 g for 5 min, the pellets were resuspended in 100 µL of SB++ buffer (180 mM Tris-HCl pH 6.8, 6.7 M Urea, 4.2% SDS, 80 µM EDTA, 1.5% v/v Beta-mercaptoethanol, 12.5 µM Bromophenol blue). Denaturation was performed by heating 5 min at 65°C. Pre-cleared extracts were resolved on 12% precast polyacrylamide gel (Bio-Rad, cat. 4561043) and blotted on a PVDF membrane (GE Healthcare, cat. 10600023). Membranes were probed with mouse anti-AID antibody diluted at 1:1000 (CliniSciences, M214-3), or anti-GAPDH antibody diluted at 1:10000 (Invitrogen, MA515738), and revealed with an HRP-conjugated anti-mouse IgG antibody diluted at 1:10000 (Abcam, ab6789) using Immobilon Forte western HRP substrate (Merck, WBLUF0100) and a Chemidoc MP Imaging system (BioRad).

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## Hi-C read alignment and generation of contact maps

16

Paired-end reads are aligned and processed using Hicstuff pipeline function 102 with the following options: -m cutsite -e DpnII,HinfI -q 20 -n -p -d -f -F -D -o "output folder". Briefly, pairs of reads are aligned iteratively and independently using Bowtie2 in its most sensitive mode to the S. cerevisiae reference genome R64-2-1 deleted for the HML, MAT, HMR, URA3, and LYS2 sequences (S288c_DSB). Each uniquely mapped read with a quality equal or greater to 20 using the -q 20 option was assigned to a restriction fragment generated from the genome fasta file with the -e DpnII,HinfI option. Spurious contacts (i.e. uncuts and loops) were filtered as described in ref. 103 using the -f option and the distribution of retained events plotted using the -p option. PCR duplicates discarded using the -D option. The contact probability as function of the genomic distance was computed for whole chromosomes using the -d option. All intermediate alignment and pair files were kept using the -n option.

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27

## ssHi-C read alignment

28

The ssHi-C alignment pipeline is depicted in **Figure S1B**. First, the reference genome is edited so as to introduce the sequence of the annealing oligonucleotides (or its reverse-complement) in place of the

29
endogenous matching sequence on the Watson strand. The endogenous sequences are concatenated as an artificial "trash" chromosome (S288c_DSB_LY_Capture_artificial). This modified genome sequence containing both the original reference and the modified sequence is used for competitive mapping of the reads. Reads originating from ligation events made by the fragment in a ssDNA form will map at the SNP-containing fragment, while reads originating from ligation events made by the fragment in a dsDNA form will map on the unmodified sequence on the "trash" chromosome. Read mapping is conducted as for Hi-C using the Hicstuff *pipeline* function. The sshicstuff *pipeline* function was run with the following options: -b 1000 -b 2000 -b 5000 -b 10000 -F -I -L -N -n 2 -E chr2 -E chr3 -E chr5 -E 2_micron -E mitochondrion -E chr_artificial_donor -E chr_artificial_ssDNA --binning-aggregate-cen 10000 --binning-aggregate-telo 1000 --window-size-cen 150000 --window-size-telo 15000 -o output folder --copy-inputs. This pipeline isolates contacts made by ssDNA fragments to generate "4C-like" plots in unbinned format or binned at various resolution (-b), computes statistics, determines the absolute number of unique contacts per fragment and generates aggregated plots around centromeres and telomeres, except of excluded chromosomes (-E).

## Generation of ratio maps

Ratio maps were generated from sparse matrices using Hicstuff *view* function, normalized (SCN normalization) ¹⁰³, log-transformed and binned. Alternatively, ratio maps were generated with Serpentine ¹⁰⁴. Briefly, two matrices binned at 1 kb of the chromosome of interest were subsampled to contain the same number of contacts. Comparison of contact maps was performed using the default threshold parameters (50 and 10) over 30 cycles. The detrending constant was set to 0.

## Generation of aggregated contact maps

Intra- and inter-chromosomal centromere pile-up contact maps were generated with Chromosight *quantify* with default parameters ¹⁰⁵.

## Computation of the contact probability as a function of genomic distance

Computation of the contact probability as a function of genomic distance P_c(s) and its derivative have been determined using Hicstuff *distance law* function with default parameters, averaging the contact data of entire chromosome. The mitochondrial genome, 2µ plasmid and artificial chromosomes were excluded from the analysis.
1 **Quantification of coverage from Hi-C data**

2 Quantification of coverage from *bam* alignment files generated with the Hicstuff *pipeline* function,
3 which uses bowtie2 in unpaired mate mode, were sorted and merged with samtools *sort* and *merged*
4 function. The coverage was computed over 500 bp windows without overlap using Tinycov *covplot*
5 function. The absolute coverage per bin was normalized onto the median coverage, and divided over
6 that of the average of undamaged samples (AD161, AD265, AD266).

7 **Quantification and statistical analysis**

8 Statistical analysis

9 The proportion of intra-chromosomal contacts were compared using a non-parametric Mann-Whitney
10 Wilcoxon rank sum test, two-tailed. Statistical cut-off was set at 0.05. P-values below this cut-off are
11 denoted by a star (*) in figures. Statistical tests were performed under R 3.6.2. Linear regression and
12 data smoothing over 4, 10 or 30 kb sliding windows was performed under Graphpad Prism 9. No
13 statistical method was used to predetermine sample size. Individual ssDNA sites that yielded less than
14 20 unique contacts were removed from the analyses. No other data were excluded from the analyses.
15 The experiments were not randomized. The investigators were not blinded to allocation during
16 experiments and outcome assessment.

17 The number of times an experiment has been repeated and the nature of the data representation (mean,
18 SEM, etc.) is indicated in the figure legends.

19 Additional resources

20 Gaussian contact probability model

21 Briefly, we modeled the NPF as a homogeneous semi-flexible chain characterized by a persistence
22 length Lp and a contour length Lc. Lp corresponds to the typical lineic distance along the chain below
23 which the NPF could be considered as rigid, and Lc to the lineic distance along the chain between the
24 resection front and the DSB. Since the overall genome organization is not significantly affected during
25 DNA repair compared to normal G2/M, we posited that the probability for a ssDNA probe to contact
26 a dsDNA fragment depends on the relative 3D position of the resection front compared to the dsDNA
27 locus in normal condition (no break) and on the probability of the ssDNA probe inside the NPF
28 emanating from the resection front to be found in close proximity to the dsDNA. The NPF thus plays
the role of a fishing rod in the hands of a fisherman located at the resection front with a hook attached at the probe position (Figure 2H of the main text). Assuming that the distribution of 3D relative positions between any pairs of loci to be Gaussian<sup>106</sup>, we could then express the average contact probability between any ssDNA probe with any dsDNA fragment as a function of the contact probability in normal conditions, of the statistics of position of the resection front and of the spatial organization of the NPF (see Supplementary Code 1). The contact probability in normal conditions is given by Hi-C experiments. The statistics of resection front is extracted from a simple 1D stochastic model of the resection front around the DSB and fitted on the experimental Hi-C coverage data in DSB conditions. The spatial properties of the NPF are given assuming a worm-like-chain statistics depending on $L_p$ and $L_c$. The only unknown parameter in the model is $L_p$ that we then varied to fit the experimentally-measured fraction of *trans* contact. All the mathematical details of the model are given in the Supplementary Code 1.

## Software availability

ssHicstuff<sup>107</sup> (version 1.0 available at https://github.com/Piazzalab/sshicstuff)

Hicstuff<sup>102</sup> (version 3.1.2 available at https://github.com/koszullab/hicstuff)

Chromosight<sup>105</sup> (version 1.6.3, available at https://github.com/koszullab/chromosight)

Serpentine<sup>104</sup> (version 0.1.3, available at https://github.com/koszullab/serpentine)

Tinycov (version 0.3.1, available at https://github.com/cmdoret/tinycov)

TinyMapper (version 0.1, available at https://github.com/js2264/tinyMapper)

R (version x64 3.6.2 is available online at https://cloud.r-project.org/)

Bowtie2<sup>108</sup> (version 2.3.5.1 available online at http://bowtie-bio.sourceforge.net/bowtie2/)

Samtools<sup>109</sup> (version 1.3.1 available online at https://github.com/samtools/samtools)

Bedtools<sup>110</sup> (version 2.27.1 available online at https://github.com/arq5x/bedtools2)

Cooler<sup>111</sup> (version 0.9.1 available online at https://github.com/open2c/cooler)

Flowing Software (version 2.5.1 available online at https://flowingsoftware.com/download/)

Custom scripts and reference genomes used in this study are available at Github

https://github.com/Piazzalab/ssHiC_MolCell_2024 under Zenodo release
1 https://doi.org/10.5281/zenodo.13149251. Original western blot images are available at Mendeley

2 Data at https://data.mendeley.com/datasets/mc3vvgb8pc/1

3 **Supplemental Table Titles**

4 Table S1: Genotype of the haploid *Saccharomyces cerevisiae* strains used in this study (related to

5 STAR Methods).

6 Table S2: DNA oligonucleotides used for genome editing (related to STAR Methods).

7 Table S3: Annealing oligonucleotides used for ssHi-C (related to STAR Methods and Figure 1).

8 Table S4: Capture oligonucleotides used for ssHi-C (related to STAR Methods and Figure 1).

9 Table S5: Hi-C and ssHi-C generated in this study, and corresponding figures (related to STAR

10 Methods).

11 **Supplemental item titles**

12 Code S1: Gaussian polymer model of ssDNA and stochastic model of resection (related to STAR

13 Methods and Figure 2). Annotated Python code provided as a Jupyter notebook.

14 Data S1: Input data for Code S1 (related to Figure 2).
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105. Matthey-Doret, C., Baudry, L., Breuer, A., Montagne, R., Guiglielmoni, N., Scolari, V., Jean,

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E., Campeas, A., Chanut, P.H., Oriol, E., et al. (2020). Computer vision for pattern detection in

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chromosome contact maps. Nat. Commun. 11, 5795. https://doi.org/10.1038/s41467-020-19562-

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7.

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106. Cattoni, D.I., Cardozo Gizzi, A.M., Georgieva, M., Di Stefano, M., Valeri, A., Chamousset,

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D., Houbron, C., Déjardin, S., Fiche, J.-B., González, I., et al. (2017). Single-cell absolute contact

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probability detection reveals chromosomes are organized by multiple low-frequency yet specific

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interactions. Nat. Commun. 8, 1753. https://doi.org/10.1038/s41467-017-01962-x.

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107. Mendiboure, N., and Piazza, A. (2024). Piazzalab/sshicstuff: sshicstuff package. Version v1.0

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(Zenodo). https://doi.org/10.5281/ZENODO.13149287

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https://doi.org/10.5281/ZENODO.13149287.

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108. Langmead, B., and Salzberg, S.L. (2012). Fast gapped-read alignment with Bowtie 2. Nat.

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Methods 9, 357–359. https://doi.org/10.1038/nmeth.1923.

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109. Danecek, P., Bonfield, J.K., Liddle, J., Marshall, J., Ohan, V., Pollard, M.O., Whitwham, A.,

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Keane, T., McCarthy, S.A., Davies, R.M., et al. (2021). Twelve years of SAMtools and BCFtools.

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GigaScience 10, giab008. https://doi.org/10.1093/gigascience/giab008.

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110. Quinlan, A.R., and Hall, I.M. (2010). BEDTools: a flexible suite of utilities for comparing

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genomic features. Bioinforma. Oxf. Engl. 26, 841–842.

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111. Abdennur, N., and Mirny, L.A. (2020). Cooler: scalable storage for Hi-C data and other

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<figure><img src="image_2.png" /><figcaption>A</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>G</figcaption></figure>

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<figure><img src="image_23.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_24.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_25.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_26.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_27.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_28.png" /><figcaption>D</figcaption></figure>

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<figure><img src="image_37.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_38.png" /><figcaption>E</figcaption></figure>

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<figure><img src="image_40.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_41.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_42.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_43.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_44.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_45.png" /><figcaption>I</figcaption></figure>

<figure><img src="image_46.png" /></figure>
# Supplemental information

## Author list

Agnès Dumont¹, Nicolas Mendiboure¹, Jérôme Savocco¹, Loqmen Anani¹, Pierrick Moreau², Agnès Thierry², Laurent Modolo¹, Daniel Jost¹, Aurèle Piazza¹,*

## Affiliations

¹ Université de Lyon, ENS de Lyon, Université Claude Bernard, CNRS UMR5239, Laboratoire de Biologie et Modélisation de la Cellule, 46 Allée d'Italie, 69007 Lyon, France

² Unité Régulation spatiale des génomes, Institut Pasteur, CNRS UMR3525, 75015 Paris, France

* Corresponding author: aurele.piazza@ens-lyon.fr
Supplementary figures and table legends

<figure><img src="image_47.png" /></figure>

<figure><img src="image_48.png" /></figure>
**Figure S1: Rationale of ssHi-C, oligonucleotides design and reads assignment. (Related to Figure 1)**

(A) Example of annealing and capture oligonucleotide design for a DpnII site located 2599 bp away from the HO cut-site (L2599 in **Figure 1E**).

(B) Pipeline for alignment of ssHi-C reads and filtering and analysis of ssDNA contacts. The reference genome is modified so as to introduce the sequence of the annealing oligonucleotides. This modified genome sequence containing both the original reference and the modified sequence is used for competitive mapping of the reads. Reads originating from ligation events made by the fragment in a ssDNA form will map at the SNP-containing fragment, while reads originating from ligation events made by the fragment in a dsDNA form will map on the unmodified sequence. In the case of MfeI/SspI digestion, no reads map at the unmodified sequences (see **Figure S2B**).

(C) Location of the captured ssDNA and dsDNA sites relative to the centromere and telomere. The *ARG4* dsDNA control is located at an equivalent distance from a centromere as the ssDNA sites.
<figure><img src="image_49.png" /><figcaption>Figure S2: ssHi-C detects both ssDNA and dsDNA contacts. (Related to Figure 1)</figcaption></figure>

(A) Representative read coverage of ssHi-C libraries obtained in WT cells (APY266) 4h post-DSB induction with annealing oligo mixes targeting ssDNA either on both sides of the DSB (All) or only the left or right ends. A ssHi-C library containing all annealing oligonucleotides but lacking endogenous ssDNA (No DSB; APY142) does not exhibit read enrichment at the target sites. The capture oligonucleotide mix was kept constant in all conditions.
(B) Left: Representative Hi-C and ssHi-C results in WT cells (APY266) 4h post-DSB induction, or in cells lacking the DSB-inducible construct. Restoration of restriction sites is DSB-dependent, and their enrichment depends on the presence of capture oligonucleotides. ssDNA sites restoration is partial and varies in efficiency from site to site (black arrows). Right: A representative control dsDNA site on chr. XV is efficiently captured with and without DSB.

(C) Targeted fragment enrichment in annealing oligonucleotide dropout experiments relative to ssHi-C library containing all annealing oligonucleotides. The bottom plot is the average of data in the upper plots.

(D) dsDNA and ssDNA sites enrichment over genome average in the presence and absence of a DSB. Each dot represents an independent site.

(E) ssDNA sites enrichment over genome average at a distance from the DSB site at 2 and 4 hours post-DSB induction, or in the absence of DSB. Dotted lines: position at which 50% of molecules have been resected at 2 and 4 hours post-DSB induction. The extent of resection partly accounts for the heterogeneity of ssDNA enrichment.

(F) Hi-C and ssHi-C contact maps of chr. V in WT cells 4 hours post-DSB induction, revealing ssDNA-specific contact stripes emanating from the DSB region and spreading along the chromosome. Same sample as in **Figure 1F**. Hi-C and ssHi-C coverage is shown. Bin: 1 kb.

(G) Comparison of the $P_c(s)$ and its derivative obtained from Hi-C and corresponding ssHi-C libraries of WT cells 4 hours post-DSB induction. Data represent mean ± SEM of 4 biological replicates.

(H) Comparison of the *trans* contacts for each chromosome obtained from Hi-C and corresponding ssHi-C libraries of WT cells 4 hours post-DSB induction. Data are average from 4 biological replicates. Red bar: median.

(I) Comparison of proportion of *trans* contacts determined in WT cells 2 and 4 hours post DSB-induction at the 7 captured dsDNA sites (ssHi-C, left) or averaged over the genome (Hi-C, right). Data represent mean ± SEM of 4 biological replicates.

(J) 4C-like representation of the distribution of contacts by the *ARG4* locus before capture enrichment (Hi-C; left) and after capture enrichment (ssHi-C; right). Data smoothed over 4 kb.
<figure><img src="image_50.png" /></figure>

<figure><img src="image_51.png" /></figure>

<figure><img src="image_52.png" /></figure>

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<figure><img src="image_60.png" /></figure>

<figure><img src="image_61.png" /></figure>

<figure><img src="image_62.png" /><figcaption>Figure S3: ssDNA contacts are constrained by nearby centromere anchoring at the SPB, but to a lower extent than DSB-flanking dsDNA sites. (Related to Figure 2).</figcaption></figure>
(A) Proportion of *trans* contacts determined by Hi-C along chr. V (right) and averaged over the genome (left) in WT cells without DSB arrested for 4 hours in G1 with alpha-factor and in metaphase upon repression of *CDC20* expression ¹, and in WT and *rad51Δ* cells 4 hours post-DSB induction (n = 2, 2, 4, and 2 biological replicates in APY266, APY537, APY266 and APY679, respectively). A partly Rad51-independent increase in *trans* contacts is observed in the ~30 kb DSB-surrounding region, to levels observed in undamaged G1 cells.

(B) Same data as in (A) with the addition of *trans* contact profiles at 2 hours post-DSB induction, and Hi-C coverage in WT and *rad51Δ* cells. The region exhibiting Rad51-independent increase in *trans* contacts extends over to the lowest Hi-C coverage section (~10 kb at 4 hours), which corresponds to a flat unresected region. Reads in this unresected regions likely originates from a minor fraction of the cell population (<5 %) still in G1 (Figure 1B). Consequently, Hi-C contact data in the ~10 kb surrounding the DSB region are dominated by this unresected G1 sub-population. It explains why the proportion of *trans* contacts is identical to a G1-arrested population, and independent on Rad51.

(C) Zoom of the Hi-C and ssHi-C data in Figure 2A around the DSB region. Data smoothed over 4 kb.

(D) Contact distribution in the DSB region for individual ssDNA sites. Data from the same samples as in D, smoothed over 4 kb.

(E) Contact distribution in the DSB region for the L8073 ssDNA site, located in the vicinity and at ~8 kb from the resection front at 2 and 4 hours post-DSB induction, respectively.

(F) Frequency of *trans* contacts with centromere-surrounding regions for the left and right DSB ends and the control (ARG4) dsDNA site in WT cells 2 and 4 hours post-DSB induction, determined by Hi-C and ssHi-C. The Hi-C viewpoint corresponds to a 5 kb bin located 5-10 kb away from the DSB site. Data represent mean ± SEM of 4 biological replicates.

(G) Among *trans* contacts only, average contact frequency between ssDNA sites and 30 kb telomeric regions as a function of chromosome arm length in WT cells. Data show mean ± SEM of 4 biological replicates. Linear regressions for the left and right ssDNA sites are shown.

(H) Average genome-wide contact distribution of ssDNA sites on the left and right of the DSB, and for a control dsDNA site located at the same distance from a centromere as the DSB site 4h post-DSB induction in untreated (n = 4) or nocodazole-treated (n = 1) WT cells (APY266). Anchors: viewpoints. Orange arrow: donor. Dotted lines: centromeres. Data smoothed over 30 kb.

(I) Aggregated ratio maps of 29 kb windows centered on the 16 centromeres over randomly chosen positions on the diagonal, from Hi-C data 4 hours post-DSB induction in untreated and nocodazole-treated WT cells.
(J) Contact frequency between all pairs of centromeres, from Hi-C data 2 and 4 hours post-DSB induction in untreated and nocodazole-treated WT cells. Median and interquartile range is shown.

(K) Same as (G) for nocodazole-treated WT cells.

(L) Average contact frequency with *trans* centromere-surrounding regions for the left and right ssDNA ends and the control (*ARG4*) dsDNA site in 4 hours post-DSB induction. From data in (H).

(M) Average contact frequency with *trans* centromeres (1 kb bin) for each ssDNA sites, as a function of the distance to the DSB sites. From data in (H). Error bars show SEM.
<figure><img src="image_63.png" /><figcaption>Figure S4: Rad51 promotes genome-wide homology search by DSB-proximal ssDNA sites. (related to Figure 3)</figcaption></figure>

(A) Representative Hi-C contact maps and Serpentine ratio maps of chr. V in WT (APY266, n = 4) and *rad51Δ* (APY679) cells 2 and 4 hours post-DSB induction.

(B) Ratio maps of the 100 kb region surrounding the DSB site of the data in (A). Bin: 2 kb.

(C) FACS profiles of WT (APY266) and *rad51Δ* cells (APY679).

(D) $P_c(s)$ and derivatives of WT and *rad51Δ* Hi-C data in (A). Data: mean ± SEM of 4 and 2 biological replicates, respectively.
(E) *Trans* contact frequency determined from WT and *rad51Δ* Hi-C data (n =4 and 2 biological replicates, respectively). Each dot represents a chromosome. Bar: Median. Error bars: interquartile range.

(F) Average genome-wide contact distribution of the left and right DSB ends 2 hours post-DSB induction in WT cells (APY266) and *rad51Δ* cells (APY679; pondered). Other legends as in **Figure 2A**.

(G) Zoom of the ssHi-C data in (F) around the DSB region, smoothed over 4 kb.

(H) Pondered contact distribution in the DSB region for individual ssDNA sites on the left (top) and right (bottom) DSB ends. Data from the same samples as in (F-G), smoothed over 4 kb. Dotted lines represent the position of individual viewpoints.

(I) Contact frequency of the average left ssDNA, right ssDNA, and of a control dsDNA site (*ARG4*) with *trans* centromere-flanking regions of data in **Figure 3B**. Top: frequency of total contacts. Bottom: Fraction of *trans* contacts only. Data: mean ± SEM. Dotted line: genome average.

(J) Among *trans* contacts only, average contact frequency between DSB-proximal ssDNA sites and 30 kb telomeric regions as a function of chromosome arm length in *rad51Δ* cells 4 hours post-DSB induction. Data show mean ± SEM of 2 biological replicates. Dotted line indicates the genome average.

WT data are from **Figure S3J**.
<figure><img src="image_64.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_65.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_66.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_67.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_68.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_69.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_70.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_71.png" /><figcaption>I</figcaption></figure>

<figure><img src="image_72.png" /><figcaption>J</figcaption></figure>

<figure><img src="image_73.png" /><figcaption>K</figcaption></figure>

<figure><img src="image_74.png" /></figure>
**Figure S5: Exo1 catalytic activity promotes genome-wide homology search. (Related to Figure 4)**

(A) Hi-C contact maps and Serpentine ratio maps of chr. V in WT (APY266, n = 4), *exo1Δ* (APY1160, n = 1) and *exo1-D173A* (*exo1-cd*, APY536, n = 1) cells 2 and 4 hours post-DSB induction.

(B) Ratio maps of the 100 kb region surrounding the DSB site of the data in (A). Bin: 2 kb.

(C) FACS profiles of WT (APY266), *exo1Δ* (APY1160) and *exo1-D173A* (*exo1-cd*, APY536) cells.

(D) $P_c(s)$ and derivatives of data in (A).

(E) *Trans* contact frequency determined from WT, *exo1Δ* and *exo1-cd* Hi-C data in (A). Each dot represents a chromosome. Bar: Median. Error bars: interquartile range.

(F) Ratio of *trans* contact frequency in Exo1-deficient cells over WT cells along chr. V at 4 hours post-DSB induction. Data represent mean ± SEM. Bin: 2 kb.

(G) Ratio maps of Exo1-deficient cells over WT cells of chr. V with the rest of the genome.

(H) Contact frequency of the DSB-proximal (< 8 kb from DSB) left ssDNA, right ssDNA, and of a control dsDNA site (*ARG4*) with *trans* centromere-flanking regions, from data of **Figure 4C**. Top: frequency of total contacts. Bottom: Fraction of *trans* contacts only. Data: mean ± SEM. Dotted line: genome average.

(I) Among *trans* contacts only, average contact frequency between DSB-proximal ssDNA sites and 30 kb telomeric regions as a function of chromosome arm length in Exo1-deficient cells 4 hours post-DSB induction. Data show mean ± SEM of 2 biological replicates. Dotted line indicates the genome average. WT data are from **Figure S3J**.

(J) Contact distribution in the DSB region for individual ssDNA sites on the left (top) and right (bottom) DSB ends in Exo1-deficient cells at 4 hours post-DSB induction. Dotted lines represent the position of individual viewpoints.

(K) *Trans* contact frequency for individual ssDNA sites on the left and right ssDNA ends as a function of the distance to the corresponding resection front in WT and Exo1-deficient cells at 2 and 4 hours post-DSB induction. Data: mean ± SEM. Dotted lines: baseline *trans* contact frequency of the dsDNA regions surrounding the resection front, lower for the detached left chromosomal fragment in *Exo1*-deficient cells, see (F-G).
<figure><img src="image_75.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_76.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_77.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_78.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_79.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_80.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_81.png" /><figcaption>J</figcaption></figure>

<figure><img src="image_82.png" /><figcaption>K</figcaption></figure>

<figure><img src="image_83.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_84.png" /><figcaption>I</figcaption></figure>

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**Figure S6: Cohesin directs homology search by DSB-distal ssDNA sites in cis. (Related to Figure 5)**

(A) Hi-C contact maps and Serpentine ratio maps of chr. V in WT (APY266, n = 4), Scc1-depleted (APY1481, n = 1) and Scc1-depleted *rad51Δ* (APY1500, n = 1) cells at the indicated time post-DSB induction.

(B) FACS profiles of WT, Scc1-depleted (APY1481) and Scc1-depleted *rad51Δ* (APY1500) cells.

(C) $P_c(s)$ and derivatives of data in (A).

(D) *Trans* contact frequency determined from WT, Scc1-depleted and Scc1-depleted *rad51Δ* Hi-C data in (A). Each dot represents a chromosome. Bar: Median. Error bars: interquartile range.

(C-D) Undamaged G1-arrested (APY266, n = 1) and metaphase-arrested (cdc20-repressed; APY537, n = 2) cells are shown for comparison.

(E-F) Average genome-wide contact distribution of the left and right ssDNA ends at 2 (left) and 4 (right) hours post-DSB induction in WT (APY266) and Scc1-depleted (APY1481) cells. Other legends as in **Figure 2A**.

(G) Hi-C coverage and amount of dsDNA (left) and ssDNA (right) contacts retrieved in Scc1-depleted and Scc1-depleted *rad51Δ* cells relative to WT cells. Data: mean of individual captured ssDNA and dsDNA sites.

(H) LOWESS regression of *cis* contacts made by individual ssDNA sites on the left DSB end compared to the genome-averaged $P_c(s)$ in Scc1-depleted and Scc1-depleted *rad51Δ* cells 4 hours post-DSB induction.

(I) Side-specific *cis* contact profiles of individual ssDNA sites close to the resection front in WT and Scc1-depleted cells 2 hours post-DSB induction. Other legends as in **Figure 5D**.

(J) Contact profiles of the R12113 ssDNA site in WT and Scc1-depleted cells at 4 hours post-DSB induction on both side of the DSB region.

(K) Contact profiles of the average of 3 DSB-proximal ssDNA sites on the left DSB end in WT and Scc1-depleted cells 4 hours post-DSB induction.
<figure><img src="image_86.png" /></figure>

<figure><img src="image_87.png" /></figure>

<figure><img src="image_88.png" /></figure>

<figure><img src="image_89.png" /></figure>

<figure><img src="image_90.png" /></figure>

<figure><img src="image_91.png" /><figcaption>**Figure S7: The recombination enhancer stimulates early *trans* search on chr. III.**
(Related to Figure 6)</figcaption></figure>

(A) Enrichment of *trans* contacts per chromosome, normalized on their length, for two CEN-proximal and two CEN-distal dsDNA control sites (left) and individual ssDNA sites (right) at 2 and 4 hours post-DSB induction in WT cells. Data show mean ± SEM of 4 biological replicates. Dotted line: no enrichment. A subset of six chromosomes, including the three shortest chromosomes, is shown.

(B) Same as in nocodazole-treated WT cells 2 hours post-DSB induction (n = 1).

(C) Pondered ssHi-C contact profiles on chr. III in *rad51Δ* cells 4 hours post-DSB induction.

(D) ssHi-C contact profiles on chr. III in Scc1-depleted cells 2 hours post-DSB induction.

(E) DSB-proximal (< 4 kb from the DSB) ssHi-C contact profiles on chr. III in Exo1-deficient cells 2 and 4 hours post-DSB induction.

(F) Hi-C contact profiles of DSB-flanking dsDNA regions 5 kb away to the left and right of the DSB site in WT and Exo1-deficient cells.
<figure><img src="image_92.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_93.png" /></figure>

<figure><img src="image_94.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_95.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_96.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_97.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_98.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_99.png" /><figcaption>J</figcaption></figure>

<figure><img src="image_100.png" /><figcaption>K</figcaption></figure>

<figure><img src="image_101.png" /></figure>
**Figure S8: Coordinated homology search by both DSB ends and side-specific NPF collapse in a donor-dependent manner. (Related to Figure 7)**

(A) ssHi-C contact profiles on chr. II in WT cells (APY266, n = 4), rad51Δ cells (APY679, n = 2), and exo1-D173A cells (APY536, n = 1) containing a donor at position 468 kb on chr. II and WT cells lacking a donor (APY358, n = 1) at 2 and/or 4 hours post-DSB induction.

(B) Inter-chromosomal Hi-C contact map between the DSB and the donor sites (left, bin = 4 kb) and corresponding Hi-C contact profiles of DSB-flanking dsDNA regions (right) in the same strains as in (A).

(C) Comparison of Hi-C and ssHi-C contact profiles by dsDNA regions and ssDNA sites at increasing distances from the left DSB end. The location of the viewpoints is depicted on the top left corner.

(D) Depletion procedure and Western blot analysis of Pol3-iAID alone or in combination with Rfc1-AID.

(E) FACS profiles of Pol3-depleted (APY1352) and Pol3- and Rfc1-depleted (APY1350) cells.

(F) Hi-C coverage in the DSB-surrounding region 4 hours post-DSB induction in WT, Pol3-depleted and Pol3/Rfc1-depleted cells.

(G) Hi-C contact maps of chr. V in WT (APY266, n = 4), Pol3-depleted (APY1352, n = 1) and Pol3/Rfc1-depleted (APY1350, n = 1) cells 4 hours post-DSB induction.

(H) $P_c(s)$ and its derivatives of data in (F).

(I) *Trans* contact frequency for individual chromosomes of data in (F).

(J) Inter-chromosomal Hi-C contact map between the DSB and the donor sites in WT (APY266), Pol3-depleted (APY1352) and Pol3- and Rfc1-depleted (APY1350) cells 4 hours post-DSB induction. Bin: 4 kb. Arrow: contact enrichment in the direction of D-loop extension lost in Pol3/Rfc1-depleted cells.

(K) Ratio map of chr. V in Pol3-depleted (top) and Pol3- and Rfc1-depleted (bottom) over WT cells.
# KEY RESOURCES TABLE

## Key resources table

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3">Antibodies</td></tr><tr><td>Mouse anti-miniAID primary antibody</td><td>MBL</td><td>Cat#M214-3</td></tr><tr><td>Mouse anti-GAPDH primary antibody, monoclonal; clone GA1R</td><td>Invitrogen</td><td>RRID: AB_10977387</td></tr><tr><td>Goat anti-mouse immunoglobulin HRP-conjugated secondary antibody</td><td>Agilent</td><td>Cat#P0447</td></tr><tr><td colspan="3">Bacterial and virus strains</td></tr><tr><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td></tr><tr><td>Biological samples</td><td></td><td></td></tr><tr><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td></tr><tr><td colspan="3">Chemicals, peptides, and recombinant proteins</td></tr><tr><td>Zymolyaze 100T</td><td>Carl Roth</td><td>Cat#9329</td></tr><tr><td>Formaldehyde</td><td>Sigma-Aldrich</td><td>Cat#F8775</td></tr><tr><td>Ethylene glycol bis(succinimidyl succinate) (=EGS)</td><td>Fisher Scientific</td><td>Cat#10350924</td></tr><tr><td colspan="3">Critical commercial assays</td></tr><tr><td>Arima Genome-Wide HiC+ Kit</td><td>Arima Genomics</td><td>Cat#A410079</td></tr><tr><td>Qubit DNA high sensitivity kit</td><td>Thermo Scientific</td><td>Cat#Q32851</td></tr><tr><td>Colibri ES DNA Library Prep Kit for Illumina Systems with UD indexes</td><td>Thermofisher</td><td>Cat#A38606024</td></tr><tr><td>AMPure XP beads</td><td>Beckman-Coulter</td><td>Cat#A63881</td></tr><tr><td colspan="3">Deposited data</td></tr><tr><td>Raw Illumina paired-end sequencing reads</td><td>This study, Table S5</td><td>SRA accession PRJNA1139728</td></tr><tr><td>HiC in cooler format as well as unfiltered and filtered ssHiC contact data in .txt and .tsv format</td><td>This study, Table S5</td><td>GEO accession GSE272969</td></tr></tbody></table>
<table><tr><td><i>Saccharomyces cerevisiae</i> S288c R64-2-1 reference genome</td><td><i>Saccharomyces</i> genome database (SGD)</td><td>http://sgd-archive.yeastgenome.org/sequence/S288C_reference/genome_releases/S288C_reference_genome_R64-2_1_20150113.tgz</td></tr><tr><td>Original code and modified <i>Saccharomyces cerevisiae</i> reference genomes</td><td>https://zenodo.org/doi/10.5281/zenodo.13149250</td><td>https://github.com/Piazzalab/ssHiC_MoICell_2024</td></tr><tr><td>Original western blot images</td><td>Mendeley Data, doi: 10.17632/mc3vvgb8pc.1</td><td>https://data.mendeley.com/datasets/mc3vvgb8pc/1</td></tr></table>

Experimental models: Cell lines

<table><tr><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td></tr></table>

Experimental models: Organisms/strains

<table><tr><td><i>S. cerevisiae</i> strain background W303</td><td>ATCC</td><td>ATTC: 208352</td></tr><tr><td><i>S. cerevisiae</i> strains generated and/or used</td><td>This study</td><td>Table S1</td></tr><tr><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td></tr></table>

Oligonucleotides

<table><tr><td>Oligonucleotides used for genome editing</td><td>This study</td><td>Table S2</td></tr><tr><td>Annealing oligonucleotides used for ssHi-C</td><td>This study</td><td>Table S3</td></tr><tr><td>Capture oligonucleotides used for ssHi-C</td><td>This study</td><td>Table S4</td></tr><tr><td></td><td></td><td></td></tr></table>

Recombinant DNA

<table><tr><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td></tr></table>

Software and algorithms

<table><tr><td>ssHicstuff</td><td>https://doi.org/10.5281/zenodo.13149287</td><td>https://github.com/Piazzalab/sshicstuff</td></tr><tr><td>HiCstuff</td><td>https://doi.org/10.5281/zenodo.4066363</td><td>https://github.com/koSzullab/hicstuff</td></tr><tr><td>Chromosight</td><td>https://doi.org/10.1038/s41467-020-19562-7</td><td>https://github.com/koSzullab/chromosight</td></tr><tr><td>Serpentine</td><td>https://doi.org/10.1093/bioinformatics/btaa249</td><td>https://github.com/koSzullab/serpentine</td></tr><tr><td>Tinycov</td><td>Cyril Matthey-Doret, Amaury Bignaud</td><td>https://github.com/cmdoret/tinycov</td></tr></table>
<table><tr><td>TinyMapper</td><td>Jacques Serizay, Cyril Matthey-Doret</td><td>https://github.com/js2264/tinyMapper</td></tr><tr><td>R</td><td>doi:10.21105/joss.00571</td><td>https://cloud.r-project.org/</td></tr><tr><td>Bowtie2</td><td>https://doi.org/10.1038/nmeth.1923</td><td>http://bowtie-bio.sourceforge.net/bowtie2/</td></tr><tr><td>Samtools</td><td>https://doi.org/10.1093/gigascience/giab008</td><td>https://github.com/samtools/samtools</td></tr><tr><td>Cooler</td><td>https://doi.org/10.1093/bioinformatics/btz540</td><td>https://github.com/open2c/coolerv</td></tr><tr><td>Flowing Software</td><td>Perttu Terho, Cell Imaging Core, Turku Centre for Biotechnology</td><td>https://flowingsoftware.com/download/</td></tr><tr><td>Other</td><td></td><td></td></tr><tr><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td></tr></table>
<table><caption>Table S1: Genotype of the haploid *Saccharomyces cerevisiae* strains used in this study.</caption><thead><tr><th>Strain</th><th>Relevant genotype</th><th>Source</th></tr></thead><tbody><tr><td>APY266</td><td>MAT<sup>a</sup>-inc, ura3::LY-HOcs, lys2::LY, trp1::GAL-HO-hphMX, his3-11,15, can1-100, leu2-3,112, ade2-1, RAD5</td><td>Piazza et al. 2021</td></tr><tr><td>APY679</td><td>MAT<sup>a</sup>-inc, ura3::LY-HOcs, lys2::LY, trp1::GAL-HO-hphMX, his3-11,15, can1-100, leu2-3,112, ade2-1, RAD5, rad51::kanMX</td><td>This study</td></tr><tr><td>APY1160</td><td>MAT<sup>a</sup>-inc, ura3::ura3::LY*-HOcs, lys2::LY, trp1::GAL-HO-hphMX, can1-100, ade2-1, leu2-3,112, his3-11,15, RAD5, exo1::KMX (*contains mutations in the LY homology sequence)</td><td>This study</td></tr><tr><td>APY536</td><td>MAT<sup>a</sup>-inc, ura3::LY-HOcs, lys2::LY, trp1::GAL-HO-hphMX, can1-100, ade2-1, leu2-3,112, his3-11,15, RAD5, exo1-D173A::TRP1</td><td>Piazza et al. 2021</td></tr><tr><td>APY537</td><td>MAT<sup>a</sup>-inc, ura3::loxP, lys2::URA3, trp1::GAL-HO-hphMX, pMET-CDC20-TRP, his3-11,15, leu2-3,112, can1-100, ade2-1, RAD5</td><td>Piazza et al. 2021</td></tr><tr><td>APY1481</td><td>MAT<sup>a</sup>-inc, ura3::LY-HOcs, lys2::LY, trp1::GAL-HO-hphMX, his3::pADH1-OsTIR1-9Myc::HIS3, can1-100, leu2-3,112, ade2-1, RAD5, SCC1-PK3-AID::kanMX</td><td>This study</td></tr><tr><td>APY1500</td><td>MAT<sup>a</sup>-inc, ura3::LY-HOcs, lys2::LY, trp1::GAL-HO-hphMX, his3::pADH1-OsTIR1-9Myc::HIS3, can1-100, leu2-3,112, ade2-1, RAD5, SCC1-PK3-AID::kanMX, rad51::kanMX</td><td>This study</td></tr><tr><td>APY513</td><td>MAT<sup>a</sup>-inc, ura3::LY-HOcs, lys2::LY, trp1::GAL-HO-hphMX, can1-100, his3::pADH1-OsTIR1-9Myc::HIS3, ade2-1, leu2-3,112, RAD5, CDC45-FlagX5-mini-AID::kanMX</td><td>Piazza et al. 2021</td></tr><tr><td>APY1548</td><td>MAT<sup>a</sup>-inc, ura3::LY-HOcs, lys2::LY, trp1::GAL-HO-hphMX, his3-11,15, leu2-3,112, can1-100, ade2-1, RAD5, RE Δ ura3::LY-HOcs, lys2::LY, can1-100, trp1::GAL-HO-hphMX, ade2-1, his3-11,15 leu2-3,112 RAD5, chrIV-845464::RE(700 bp)</td><td>This study</td></tr><tr><td>APY1497</td><td>MAT<sup>a</sup>-inc, ura3::LY-HOcs, lys2::URA3, trp1::GAL-HO-hphMX, his3-11,15, can1-100, leu2-3,112, ade2-1, RAD5</td><td>This study</td></tr><tr><td>APY358</td><td>MAT<sup>a</sup>-inc, ura3::LY-HOcs, lys2::LY, trp1::GAL-HO-hphMX, his3-11,15, can1-100, leu2-3,112, ade2-1, RAD5, pol3-iAID (KanMX N-term &amp; hphMX C-term), RFC1-AID-9Myc-hphMX, SSN6::pST1760 (TetR, OsTir1):HIS3</td><td>This study</td></tr><tr><td>APY1350</td><td>MAT<sup>a</sup>-inc, ura3::LY-HOcs, lys2::LY, trp1::GAL-HO-hphMX, his3-11,15, can1-100, leu2-3,112, ade2-1, RAD5, pol3-iAID (KanMX N-term &amp; hphMX C-term), SSN6::pST1760 (TetR, OsTir1):HIS3</td><td>This study</td></tr><tr><td>APY1352</td><td>MAT<sup>a</sup>-inc, ura3::LY-HOcs, lys2::LY, trp1::GAL-HO-hphMX, his3-11,15, can1-100, leu2-3,112, ade2-1, RAD5, pol3-iAID (KanMX N-term &amp; hphMX C-term), SSN6::pST1760 (TetR, OsTir1):HIS3</td><td>This study</td></tr></tbody></table>
<table><caption>Table S2: Oligonucleotides used for</caption><thead><tr><th></th><th></th></tr></thead><tbody><tr><td>RE deletion (chrIII:29107-30801)</td><td></td></tr><tr><td>Cas9 guide left</td><td></td></tr><tr><td>Cas9 guide right</td><td></td></tr><tr><td>Repair template</td><td></td></tr><tr><td>RE insertion (chrIV:845464)</td><td></td></tr><tr><td>RE-atchrIV-845464-F</td><td></td></tr><tr><td>RE-atchrIV-845464-R</td><td></td></tr></tbody></table>
## genome editing

### Sequence (5'-3')

ACAACGGAATCATTGAGCGT

CAACGGAATCATTGAGCGA

AGCATCAACACATAAATCCTTGCTTAGCTCAATTAAATATACTAGTAAATAAGTATATAAACAATAATTTTGCA

TTTTATTTTACTGGAAACTCTTCTCAAAACCAAATTGCGCAAGGATTGATTCAGTACAATTATGCAAACTCGAA

AAGTAAATAAACAAAAAGATACA/CTTATAAAATGAATATTTCAATTGATGAATAGCTATATATTGGATACAAA

AATTAGCATTTAATCGAAAAACTGCAGCATGTATTTAATCGAAAACTACAGCATGTAGCTATGATACGACAGAAG

ATTTTTGTTTTTATAGTTAAGTCAAGAAGAAATTCTATTTGTCCAGCAATCCGGCGCAAAGAAGACTACTAAAG

GG

### Sequence (5'-3')

TAACTAGGATTTAATCATAAAAAAAAAAGTGATTGACACTGTTATTCAGTTCTGGACTATCGTATAGAGTTGAG

TGAAAGGTAAATAAAC

TGGGCTTGGTAATCGTTGAAAAATTTTCACATATTTTATGAACAGCTGAGCCTATTAACAATGAAAATAGACCG

AAGTACAACAATATCC
<caption>Table S3: Annealing oligonucleotides used for ssHi-C</caption>

Nucleotide in lower case mark SNPs relative to the reference sequence. DpnII sites are in bold.

<table><thead><tr><th>Name</th><th>Strand</th><th>DpnII_to_HOcs<br/>_distance</th><th>S288c_DSB_L<br/>Y_genome<br/>DpnII_site_st<br/>art</th></tr></thead><tbody><tr><td>Probe_URA-L-17213-Mfel-RC_v5</td><td>-</td><td>-17213</td><td>101550</td></tr><tr><td>Probe_URA-L-16220-Mfel-RC_v5</td><td>-</td><td>-16220</td><td>102543</td></tr><tr><td>Probe_URA-L-15683-Sspl-RC</td><td>-</td><td>-15683</td><td>103080</td></tr><tr><td>Probe_URA-L-9924-Mfel-RC_v5</td><td>-</td><td>-9924</td><td>108839</td></tr><tr><td>Probe_URA-L-6532-Mfel-RC</td><td>-</td><td>-6532</td><td>112231</td></tr><tr><td>Probe_URA-L-6065-Sspl-RC</td><td>-</td><td>-6065</td><td>112698</td></tr><tr><td>Probe_URA-L-4543-Sspl-RC</td><td>-</td><td>-4543</td><td>114220</td></tr><tr><td>Probe_URA-L-3728-Sspl-RC</td><td>-</td><td>-3728</td><td>115035</td></tr><tr><td>Probe_URA-L-3081-Mfel-RC</td><td>-</td><td>-3081</td><td>115682</td></tr><tr><td>Probe_URA-L-2599-Mfel-RC</td><td>-</td><td>-2599</td><td>116186</td></tr><tr><td>Probe_URA-R-1439-Sspl</td><td>+</td><td>1439</td><td>120223</td></tr><tr><td>Probe_URA-R-2715-93-Sspl_v5</td><td>+</td><td>2715</td><td>121510</td></tr><tr><td>Probe_URA-R-2954-Sspl_v5</td><td>+</td><td>2954</td><td>121738</td></tr><tr><td>Probe_URA-R-8073-Sspl</td><td>+</td><td>8073</td><td>126857</td></tr><tr><td>Probe_URA-R-9028-Mfel_v5</td><td>+</td><td>9028</td><td>127812</td></tr><tr><td>Probe_URA-R-10241-Sspl_v5</td><td>+</td><td>10241</td><td>129025</td></tr><tr><td>Probe_URA-R-12116-Sspl</td><td>+</td><td>12116</td><td>130900</td></tr><tr><td>Probe_URA-R-13482-Sspl_v5</td><td>+</td><td>13482</td><td>132266</td></tr><tr><td>Probe_URA-R-14040-Mfel</td><td>+</td><td>14040</td><td>132824</td></tr><tr><td>Probe_URA-L-774-Mfel-RC</td><td>-</td><td>-774</td><td>118011</td></tr><tr><td>Probe_URA-L-1560-Sspl-RC</td><td>-</td><td>-1560</td><td>117225</td></tr></tbody></table>
Annealing_oligonucleotide_sequence

ACcATACAtTTGTCTACTTTAACAATAGAAGTTtATAAGGTTGGGCCTCTTTTAAGAGaACAGGTATAGT**GATCTCATCG**
CCATGA**GATCTTGCACCTTTTcGTTATGCCTAAATTACTACAGCTaAATTGAcAATACATGTTTGTGCATTTACAGATGC
GCGAGTGATAAGAACTtTCTATTAGGTCGATTAAcCAGACATTTCTTTGTGCAACGTTGAAtTATTCCAATAG**GATCTTATGT
AAGTTT**GATCGTAGGTATAGAcTGGACAATATGCCGGAATATGTAAGGCAATTaTTCCAAGtTTTGGAAGGTATTtATTT
CGATGG**GATCTTCTTGACTTgCTTCTTCTTTGGATaCTACATTTGTGCCAtTTGTAaCGGCCTTCAATACAaCATTTGTG
TTTCAtAACTCGAGTTATTTGCGGTACCAaCATgTAAAAGAATAGAAAAtTATTCTTGTTAGAGTGtGCTCG**GATCTGTAAT
ATCAAGTCTTCGTACAATGTTAGTATAtTATTAACAATGTTTAACCATAATTCCTTGGAGAAAATTTTAG**GATCTTTGTT
ATGAAATTTCtTCTAATAGTCCTAGGAcACACATGAAGTaCTCATTTGTCAAAAtTATTGCTGTCTGTCTT**GATCGATTaA
TGTATTGTCATaAATTGGCGCAGTAGCCTCAATTTCAACGTCGTTTGcCTCTGGTGTTTGTTaATGTGCAG**GATCCATGAG
GTTAAATCCAAAACGGCAGAAGCCTGAATGAAACATATtAACCAtTTGGAGtACGTCAATaAATTCTGGG**GATCCATTGC
TTGGCA**GATCTTTAACTCGGCTTTAGTTATaCAAGTTACTTGCAtTATTCCTTCTGCGAGAGTACATTTGCCCTTAAAC
CCAATAACAtTCAGAATAcTTTTATTTTTATGTTGCAAAATAAGCTcTACGACTTTTTT**GATCTTCGACGG**GATCAGGGCC
GTAtTCATGTCAtTATTGTCAGGGGTTAACTTTCCGGTAAACTTCAAAAATaGGGACAAAAGACTAACTT**GATCCAACTC
TATCGTCATAcCTGTGCTTTCTGTTAcCGTATTGGAAtTATTCCAGCTaGtGTCGAAAaCTTCTTTCAG**GATCACGTGA
GTATTT**GATCTATTCGCAAtTTGTCGGGGCaCATTATgAATAGGTTTTGGGCACTTCGAATATAAAAGCAACTCAATGAG
ATCG**GATCCCAAATACCTACCtATAACTCAGTCTTTGAtTATTACACATTTAATTCAgTCTGCAACTAACTCTCaATC
TACTGAAAAATACGTCCGTCAaGTCTCTAGAGAGGTACTGGAAaCCcTCTTAATAcTATTACAAGCCA**GATCCACAGA
ACACAG**GATCTGAACATaAACCTGTAGAGaATGCAAGTTTGGAATTGTATAAtTATTACTCAACAtATTTTACAATTTTTA
CGTTTTTAGAATATATTGTAATAAAACACAATTaATAATACAGTTgTCTCTTCGTCCTCTtATGGTGGGAG**GATCTATTAA
ATATCATCTTGAGTAGGGACATACAtTTGGGCACCTAAAAAATAATGGTGTAAACATtTCTCTTTaAATTGG**GATCATGTGC
TGGATG**GATCGCTTAGCGCAGCAGTCAAATAgTGAGTAAATTGGTgCGCAACAATGGTTACTCTTTCATTCGAATACAGT
Original_sequence

ACGATACAATTGTCTACTTTAACAATAGAAGTTGATAAGGTTGGGCCTCTTTTAAGAGTACAGGTATAGTGATCTCATCG
CCATGA GATCTTGCACCTTTTTGTTATGCCTAAATTACTACAGCTCAATTGATAATACATGTTTGTGCATTTACAGATGC
GCGAGTGATAAGAACTATCTATTAGGTCGATTATCAGACCTTTCTTGTGCAACGTTGAAATATTCCAATAGATCTTATGT
AAGTTTGATCGTAGGTATAGACCGGACAATATGCCGGAATATGTAAGGCAATTGTTCCAAGATTTGGAAGGTATTGATTT
CGATGGGATCTTCTTGACTTCCTTCTTCTTTGGATTCTACATTTGTGCCAATTGTATCGGCCTTCAATACATCATTTGTG
TTTCAGAACTCGAGTTATTTGCGGTACCATCATCTAAAAGAATAGAAAATATTCTTGTTAGAGTGAGCTCGATCTGTAAT
ATCAAGTCTTCGTACAATGTTAGTATAATATTAACAATGTTTAACCATAATTCCTTGGAGAAAATTTTAGGATCTTTGTT
ATGAAATTTCATCTAATAGTCCTAGGACCCACATGAAGTTCTCATTTGTCAAAATATTGCTGTCTGTCTTGATCGATTCA
TGTATTGTCATCAATTGGCGCAGTAGCCTCAATTTCAACGTCGTTTGACTCTGGTGTTTGTTCCATGTGCAGATCCATGAG
GTTAAATCCAAAAACGGCAGAAGCCTGAATGAAACATATGAACCAATTGGAGGACGTCAATGAATTCTGGGGATCCATTGC
TTGGCAGATCTTTAACTCGGCTTTAGTTATCCAAGTTACTTGCAATATTTCCTTCTGCGAGAGTACATTTGCCCTTAAAC
CCAATAACAATCAGAATATTTTTATTTTTATGTTGCAAAATAAGCTGTACGACTTTTTTGATCTTCGACGGATCAGGGCC
GTAATCATGTCAATATTGTCAGGGGTTAACTTTCCGGTAAACTTCAAAAATGGGGACAAAAGACTAACTTGATCCAACTC
TATCGTCATATCTGTGCTTTCTGTTATCGTATTGGAAATATTCCAGCTCGGGTCGAAATCTTCTTTCAGGATCACGTGA
GTATTTGATCTATTCGCAATTGTCCGGGGCCCATTATCAATAGGTTTTGGGCACTTCGAATATAAAAGCAACTCAATGAG
ATCGATGATCCCAAATACCTACCAATAACTCAGTCTTTGAATATTACACATTTAATTCACTCTGCAACTAACTCTCCATC
TACTGAAAAATACGTCCGTCAGGTCTCTAGAGAGGTACTGGAACCCATCTTAATATTATTACAAAGCCAAGATCCACAGA
ACACAGGATCTGAACATCAACCTGTAGAGGATGCAAGTTTGGAATTGTATAATATTACTCAACAGATTTTACAATTTTTA
CGTTTTTAGAATATATTGTAATAAAACACAATTGATAATACAGTTCTCTCTTCGTCCTCTGATGGTGGGATCTATTAA
ATATCATCTTGAGTAGGGACATACAATTGGGCACCTAAAAATAATGGTGTAAACATATCTCTTTGAATTGGATCATGTGC
TGGATGGATCGCTTAGCGCAGCAGTCAAATATTGAGTAAATTGGTCCGCAACAATGGTTACTCTTTCATTCGAATACAGT
<table><caption>Table S4: Capture oligonucleotides used in this study</caption><thead><tr><th>Name</th><th>Orientation</th><th>Chromosome</th><th>Start_S28<br>8c_DSB_L<br>Y_Captured</th><th>End_S288<br>c_DSB_L<br>Y_Captured</th></tr></thead><tbody><tr><td>Probe_URA-L-17213-Mfel-RC</td><td>-</td><td>chr5</td><td>101554</td><td>101614</td></tr><tr><td>Probe_URA-L-16220-Mfel-RC</td><td>-</td><td>chr5</td><td>102483</td><td>102543</td></tr><tr><td>Probe_URA-L-15683-Sspl-RC</td><td>-</td><td>chr5</td><td>103084</td><td>103144</td></tr><tr><td>Probe_URA-L-9924-Mfel-RC</td><td>-</td><td>chr5</td><td>108779</td><td>108839</td></tr><tr><td>Probe_URA-L-6532-Mfel-RC</td><td>-</td><td>chr5</td><td>112171</td><td>112231</td></tr><tr><td>Probe_URA-L-6065-Sspl-RC</td><td>-</td><td>chr5</td><td>112712</td><td>112772</td></tr><tr><td>Probe_URA-L-3728-Sspl-RC</td><td>-</td><td>chr5</td><td>115039</td><td>115099</td></tr><tr><td>Probe_URA-L-3081-Mfel-RC</td><td>-</td><td>chr5</td><td>115686</td><td>115746</td></tr><tr><td>Probe_URA-L-2599-Mfel-RC</td><td>-</td><td>chr5</td><td>116200</td><td>116260</td></tr><tr><td>Probe_URA-R-1439-Sspl</td><td>+</td><td>chr5</td><td>120227</td><td>120287</td></tr><tr><td>Probe_URA-R-2715-93-Sspl</td><td>+</td><td>chr5</td><td>121440</td><td>121500</td></tr><tr><td>Probe_URA-R-2954-Sspl</td><td>+</td><td>chr5</td><td>121678</td><td>121738</td></tr><tr><td>Probe_URA-R-8073-Sspl</td><td>+</td><td>chr5</td><td>126797</td><td>126857</td></tr><tr><td>Probe_URA-R-9028-Mfel</td><td>+</td><td>chr5</td><td>127816</td><td>127876</td></tr><tr><td>Probe_URA-R-10241-Sspl</td><td>+</td><td>chr5</td><td>129029</td><td>129089</td></tr><tr><td>Probe_URA-R-12116-Sspl</td><td>+</td><td>chr5</td><td>130840</td><td>130900</td></tr><tr><td>Probe_URA-R-13482-Sspl</td><td>+</td><td>chr5</td><td>132270</td><td>132330</td></tr><tr><td>Probe_URA-R-14040-Mfel</td><td>+</td><td>chr5</td><td>132754</td><td>132814</td></tr><tr><td>Probe_URA-L-1560-Sspl-RC</td><td>-</td><td>chr5</td><td>117165</td><td>117225</td></tr><tr><td>chr4-64420-CDC13</td><td>NA (dsDNA)</td><td>chr4</td><td>64420</td><td>64480</td></tr><tr><td>chr8-428677-DNA2</td><td>NA (dsDNA)</td><td>chr8</td><td>428677</td><td>428737</td></tr><tr><td>chr15-996452-MEK1</td><td>NA (dsDNA)</td><td>chr15</td><td>996452</td><td>996512</td></tr><tr><td>chr2-650593-MET8</td><td>NA (dsDNA)</td><td>chr2</td><td>650593</td><td>650653</td></tr><tr><td>chr15-620339-PDR5</td><td>NA (dsDNA)</td><td>chr15</td><td>620339</td><td>620399</td></tr><tr><td>chr10-449622-POL31</td><td>NA (dsDNA)</td><td>chr10</td><td>449622</td><td>449682</td></tr><tr><td>chr8-140404-ARG4</td><td>NA (dsDNA)</td><td>chr8</td><td>140404</td><td>140459</td></tr></tbody></table>
Capture oligo 5'-biot (60 mers)

TGTCTACTTTAACAATAGAAGTTtATAAGGTTGGGCCTCTTTTAAGAGAACAGGTATAGT
TTGCACCTTTTcGTTATGCCTAAATTACTACAGCTaAATTGAcaAATACATGTTTGTGCAT
AGAACTtTCTATTAGGTCGATTaA CAGACATTTCTTGTGCAACGTTGAAtTATTCCAATA
GTAGGTATAGAcTGGACAATATGCCGGAATATGTAAGGCAATTaTTCCAAGtTTTGAAG
TTCTTGACTTgCTTCTTCTTTGGATaCTACATTTGTGCCAtTTGT AaCGGCCTTCAATAC
TTTCAtAACTCGAGTTATTTGCGGTACCAaCATgTAAAAGAATAGAAAAtTATTCTTGTTA
tTCTAATAGTCCTAGGAcACACATGAAGT aCTCATTTGTCAAAtTATTGCTGTCTGTCTT
TaAATTGGCGCAGTAGCCTCAATTTCAACGTCGTTTGcCTCTGGTGTTTGTTaATGTGCA
ATTGACGTaCTCCAAaTGGTTaATATGTTTCATTCAGGCTTCTGCCGTTTTGGATTTAAC
TTTAACTCGGCTTTAGTTaT aCAAGTTACTTGCAtTATTTCCTTCTGCGAGAGTACATTT
CCAATAACAtTCAGAATAcTTTTATTTTTATGTTGCAAAATAAGCTcTACGACTTTTTTG
CAtTATTGTCAGGGGTTAACTTTCCGGTAAACTTCAAAAAATGGGGACAAAAAGACTAACTT
cCTGTGCTTTCTGTTAcCGTATTGGAAtTATTTCCAGCTaGtGTCGAAAaCTTCTTTCAG
TATTCGCAAtTTGTCGGGGCaCATTAtgAATAGGTTTTGGGCACTTCGAATATAAAAGCA
CCAAATACCTACCtATAACTCAGTCTTTGAtTATTACACATTTAATTCAgTCTGCAACTA
TACGTCCGTCAaGTCTCTAGAGAGGTACTGGAAaCCcTCTTAATAcTATTACAAAGCCAA
TGAACATaAACCTGTAGAgATGCAAGTTTGAATTGTATAtTATTACTCAACAtATTTT
ATATATTGTAATAAAACACAATTaATAATACAGTTgTCTCTTCGTCCTCTtATGGTGGGA
GCTTAGCGCAGCAGTCAAATAgTGAGTAAATTGGTgCGCAACAATGGTTACTCTTTCATT
CAACTCACTTGTGGATATCTTCAACAATTTAATAGAAATGAATAGAGACGAGAAAAACAG
CTGATGACTACGAAGATGTCCAAAATCCCTCTTCTACACCTATAGTCCCTAATCGACTGA
ATGAGACCGTTGTATAGCTGCAACCTTGCAACCAAAGATGATATTGAGATGGCAGGTGGC
CCGAACTGGGACCCCACGAAGAATGAAATTTACGAGTACATCCGCAGTGACTTCAAAGAC
CTAAAGAAACCAATACTTTCCAAATCTTGAAACCAATGGATGGTTGCCTAAACCCAGGTG
GGTGGTTCTGATGAAATTATGCTCGAAGATGAAAGTGGGAGAGTGCTTCTAGTGGGAGAT
AAATAATGGCTCTTTGTCTTCTTGCATGTCTTTATCATAAGTAGATGGGATACCCTTCAA
<table><caption>Table S5: Hi-C and ssHi-C generated in this study, and corresponding figures</caption><thead><tr><th>Figures</th><th>Condition</th><th>Type</th><th>Strain</th><th>Library name(s)</th><th>Genome-wide contacts</th><th>SRA bioproject</th></tr></thead><tbody><tr><td rowspan="4">1F, 2A-G, 3A-D, 4A-F, 5B-F, 6A, 6D-F, S2-8</td><td>WT, DSB 2 hr</td><td>Hi-C</td><td>APY266</td><td>AD217; AD280</td><td>30766116; 32018726</td><td>PRJNA1139728</td></tr><tr><td>WT, DSB 2 hr</td><td>ssHi-C</td><td>APY266</td><td>AD241; AD295</td><td>4140152; 5383850</td><td>PRJNA1139728</td></tr><tr><td>WT, DSB 4 hr</td><td>Hi-C</td><td>APY266</td><td>AD154; AD218; AD281</td><td>12587556; 37473168; 39643934</td><td>PRJNA1139728</td></tr><tr><td>WT, DSB 4 hr</td><td>ssHi-C</td><td>APY266</td><td>AD162; AD242; AD296; AD414</td><td>8367736; 4124468; 5068422; 3959549</td><td>PRJNA1139728</td></tr><tr><td rowspan="14">S2A-E</td><td>No DSB</td><td>Hi-C</td><td>APY142</td><td>AD161</td><td>10965620</td><td>PRJNA1139728</td></tr><tr><td>No DSB</td><td>ssHi-C</td><td>APY142</td><td>AD169</td><td>8627340</td><td>PRJNA1139728</td></tr><tr><td>WT, DSB 4 hr, left annealing oligos only</td><td>Hi-C</td><td>APY266</td><td>AD155</td><td>11766945</td><td>PRJNA1139728</td></tr><tr><td>WT, DSB 4 hr, left annealing oligos only</td><td>ssHi-C</td><td>APY266</td><td>AD163</td><td>7411029</td><td>PRJNA1139728</td></tr><tr><td>WT, DSB 4 hr, right annealing oligos only</td><td>Hi-C</td><td>APY266</td><td>AD156</td><td>10128251</td><td>PRJNA1139728</td></tr><tr><td>WT, DSB 4 hr, right annealing oligos only</td><td>ssHi-C</td><td>APY266</td><td>AD164</td><td>8829271</td><td>PRJNA1139728</td></tr><tr><td>WT, DSB 4 hr, left DSB-proximal annealing oligos</td><td>Hi-C</td><td>APY266</td><td>AD159</td><td>11816290</td><td>PRJNA1139728</td></tr><tr><td>WT, DSB 4 hr, left DSB-proximal annealing oligos</td><td>ssHi-C</td><td>APY266</td><td>AD167</td><td>7446819</td><td>PRJNA1139728</td></tr><tr><td>WT, DSB 4 hr, left DSB-distal annealing oligos</td><td>Hi-C</td><td>APY266</td><td>AD160</td><td>10877797</td><td>PRJNA1139728</td></tr><tr><td>WT, DSB 4 hr, left DSB-distal annealing oligos</td><td>ssHi-C</td><td>APY266</td><td>AD168</td><td>7915476</td><td>PRJNA1139728</td></tr><tr><td>WT, DSB 4 hr, left staggered 1 annealing oligos</td><td>Hi-C</td><td>APY266</td><td>AD157</td><td>12354023</td><td>PRJNA1139728</td></tr><tr><td>WT, DSB 4 hr, left staggered 1 annealing oligos</td><td>ssHi-C</td><td>APY266</td><td>AD157</td><td>7606857</td><td>PRJNA1139728</td></tr><tr><td>WT, DSB 4 hr, left staggered 2 annealing oligos</td><td>Hi-C</td><td>APY266</td><td>AD158</td><td>9739763</td><td>PRJNA1139728</td></tr></tbody></table>
<table><thead><tr><th></th><th></th><th>WT, DSB 4 hr, left staggered 2 annealing oligos</th><th>ssHi-C</th><th>APY266</th><th>AD166</th><th>9029319</th><th>PRJNA1139728</th></tr></thead><tbody><tr><td rowspan="2">S3A-B, S6B</td><td>G1-arrested (α-factor)</td><td>Hi-C</td><td>APY266</td><td>HB12, HB89</td><td>23426177; 19210270</td><td>PRJNA647790</td></tr><tr><td>metaphase-arrested (cdc20)</td><td>HiC</td><td>APY537</td><td>AD265; AD266</td><td>17844910; 29231976</td><td>PRJNA1139728</td></tr><tr><td rowspan="4">S3H-M</td><td>WT, DSB 2 hr + Noco</td><td>Hi-C</td><td>APY266</td><td>AD282</td><td>29683210</td><td>PRJNA1139728</td></tr><tr><td>WT, DSB 2 hr + Noco</td><td>ssHi-C</td><td>APY266</td><td>AD297</td><td>18257038</td><td>PRJNA1139728</td></tr><tr><td>WT, DSB 4 hr + Noco</td><td>Hi-C</td><td>APY266</td><td>AD283</td><td>31547992</td><td>PRJNA1139728</td></tr><tr><td>WT, DSB 4 hr + Noco</td><td>ssHi-C</td><td>APY266</td><td>AD298</td><td>18109606</td><td>PRJNA1139728</td></tr><tr><td rowspan="4">3A-D, 6B, 7C, S3A-B, S4, S6A-C, S7C, S8A-B</td><td>rad51Δ DSB 2 hr</td><td>Hi-C</td><td>APY679</td><td>AD223; AD364</td><td>42759189; 26732781</td><td>PRJNA1139728</td></tr><tr><td>rad51Δ DSB 2 hr</td><td>ssHi-C</td><td>APY679</td><td>AD247; AD450</td><td>3885148; 21125342</td><td>PRJNA1139728</td></tr><tr><td>rad51Δ DSB 4 hr</td><td>Hi-C</td><td>APY679</td><td>AD224; AD365</td><td>30343335;29606707</td><td>PRJNA1139728</td></tr><tr><td>rad51Δ DSB 4 hr</td><td>ssHi-C</td><td>APY679</td><td>AD248; AD405</td><td>3692836; 13246255</td><td>PRJNA1139728</td></tr><tr><td rowspan="4">4A-H, 7C, S5, S7E-F, S8A-B</td><td>exo1-cd and exo1Δ DSB 2 hr</td><td>Hi-C</td><td>APY536; APY1160</td><td>AD227; AD286</td><td>27690614; 31803575</td><td>PRJNA1139728</td></tr><tr><td>exo1-cd and exo1Δ DSB 2 hr</td><td>ssHi-C</td><td>APY536; APY1160</td><td>AD235; AD301</td><td>3046064; 4750895</td><td>PRJNA1139728</td></tr><tr><td>exo1-cd and exo1Δ DSB 4 hr</td><td>Hi-C</td><td>APY536; APY1160</td><td>AD228; AD287</td><td>29535636; 29881828</td><td>PRJNA1139728</td></tr><tr><td>exo1-cd and exo1Δ DSB 4 hr</td><td>ssHi-C</td><td>APY536; APY1160</td><td>AD236; AD302</td><td>3799406; 4004171</td><td>PRJNA1139728</td></tr><tr><td rowspan="5">5B-D, S6A,C-I, S7D</td><td>Sccl1-depleted DSB 2 hr</td><td>Hi-C</td><td>APY1481</td><td>AD354</td><td>35398687</td><td>PRJNA1139728</td></tr><tr><td>Sccl1-depleted DSB 2 hr</td><td>ssHi-C</td><td>APY1481</td><td>AD401</td><td>19407040</td><td>PRJNA1139728</td></tr><tr><td>Sccl1-depleted DSB 4 hr</td><td>Hi-C</td><td>APY1481</td><td>AD355</td><td>26781764</td><td>PRJNA1139728</td></tr><tr><td>Sccl1-depleted DSB 4 hr</td><td>ssHi-C</td><td>APY1481</td><td>AD402</td><td>16628663</td><td>PRJNA1139728</td></tr><tr><td>Sccl1-depleted rad51Δ DSB 4 hr</td><td>Hi-C</td><td>APY1500</td><td>AD369</td><td>26468819</td><td>PRJNA1139728</td></tr><tr><td>5E-G</td><td>Cdc45-AID +DMSO DSB 4 hr (after G1 release)</td><td>ssHi-C</td><td>APY513</td><td>AD257; AD259</td><td>4487869; 4254821</td><td>PRJNA1139728</td></tr><tr><td rowspan="2">6C-D</td><td>Cdc45-AID +IAA DSB 4 hr (after G1 release)</td><td>ssHi-C</td><td>APY513</td><td>AD260;AD289</td><td>3749978; 3599675</td><td>PRJNA1139728</td></tr><tr><td>re Δ</td><td>ssHi-C</td><td>APY1548</td><td>AD490</td><td>13455716</td><td>PRJNA1139728</td></tr><tr><td>chrIV-845464::RE(700 bp)</td><td>ssHi-C</td><td>APY1497</td><td>AD406</td><td>13437006</td><td>PRJNA1139728</td></tr><tr><td>7B, E-G, S8H-K</td><td>WT, no donor, DSB 2 hr</td><td>Hi-C</td><td>APY358</td><td>AD362</td><td>30648544</td><td>PRJNA1139728</td></tr></tbody></table>
<table><tr><td>WT, no donor, DSB 2 hr</td><td>ssHi-C</td><td>APY358</td><td>AD403</td><td>16628663</td><td>PRJNA1139728</td></tr><tr><td>WT, no donor, DSB 4 hr</td><td>Hi-C</td><td>APY358</td><td>AD363</td><td>29990804</td><td>PRJNA1139728</td></tr><tr><td>WT, no donor, DSB 4 hr</td><td>ssHi-C</td><td>APY358</td><td>AD404</td><td>15340854</td><td>PRJNA1139728</td></tr><tr><td>Pol3-depleted, DSB 4 hr</td><td>Hi-C</td><td>APY1352</td><td>AD410</td><td>42637309</td><td>PRJNA1139728</td></tr><tr><td>Pol3-depleted, DSB 4 hr</td><td>ssHi-C</td><td>APY1352</td><td>AD479</td><td>12238915</td><td>PRJNA1139728</td></tr><tr><td>Pol3/Rfc1-depleted, DSB 4 hr</td><td>Hi-C</td><td>APY1350</td><td>AD409</td><td>25011986</td><td>PRJNA1139728</td></tr><tr><td>Pol3/Rfc1-depleted, DSB 4 hr</td><td>ssHi-C</td><td>APY1350</td><td>AD480</td><td>11005434</td><td>PRJNA1139728</td></tr></table>