DOI: 10.5061/dryad.s4mw6m9fc
title: Data from: CDCA7 is an evolutionarily conserved hemimethylated DNA sensor in eukaryotes

description: Mutations of the SNF2 family ATPase HELLS and its activator CDCA7 cause
 immunodeficiency, centromeric instability, facial anomalies (ICF)
 syndrome, characterized by DNA hypomethylation at heterochromatin. It
 remains unclear why CDCA7-HELLS is the sole nucleosome remodeling complex
 whose deficiency abrogates the maintenance of DNA methylation. We here
 identify the unique zinc-finger domain of CDCA7 as an evolutionarily
 conserved hemimethylation-sensing zinc finger (HMZF) domain. Cryo-EM
 structural analysis of the CDCA7-nucleosome complex reveals that the HMZF
 domain can recognize hemimethylated CpG in the outward-facing DNA major
 groove within the nucleosome core particle (NCP), whereas UHRF1, the
 critical activator of the maintenance methyltransferase DNMT1, cannot.
 CDCA7 recruits HELLS to hemimethylated chromatin and facilitates
 UHRF1-mediated H3 ubiquitylation associated with replication-uncoupled
 maintenance DNA methylation. We propose that the CDCA7-HELLS nucleosome
 remodeling complex assists the maintenance of DNA methylation on chromatin
 by sensing hemimethylated CpG that is otherwise inaccessible to UHRF1 and
 DNMT1.
description: **Data from: CDCA7 is an evolutionarily conserved hemimethylated DNA
 sensor in eukaryotes**
 [https://doi.org/10.5061/dryad.s4mw6m9fc](https://doi.org/10.5061/dryad.s4mw6m9fc) This submission contains the raw image data files supporting the manuscript accepted for publication in Science Advances (Wassing et al). The data include image data of SDS-PAGE and  native PAGE analyses (Coomassie Staining, Western blotting, and DNA staining). It also contains data spreadsheets (.xlsx files) for quantitation analysis. For detailed experimental procedures, materials, and the context, please read Wassing et al (2024) Science Advances (doi; 10.1126/sciadv.adp5753). **Description of the data and file structure** **Supporting files for Fig. 1A** Fig_1A.zip includes the raw tif files of all three replicates of the DNA bead pull-down experiment shown in Fig 1A-C. Magnetic beads coupled with double-stranded 54 bp DNA oligos containing unmethylated CpGs (un-Me), fully methylated CpGs (full-Me), or hemimethylated CpGs (hemi-Me; (F) and (R) to indicated 5mC in the forward- or reverse- strand, table S1), were incubated with interphase *Xenopus* egg extracts. Beads were collected after 10 min and analyzed by western blotting. SDS-PAGE was stained with SYBR Safe to visualize loading of the 54 bp DNA. For each experiment a scan of the DNA-stained gel (SYBR Safe) and Western Blot analysis are included. The data of experiment 1 (20240206_exp1) was used to assemble the representative figure displayed in Figure 1A. To generate unmethylated; fully methylated and hemimethylated biotinylated 54 bp DNA substrates (Fig. 1A-C), 54 bp DNA oligos listed in table S1 were annealed in a thermocycler and purified with size exclusion chromatography, Superdex 200 Increase 10/300 GL (Cytiva). 54 bp DNA substrates were conjugated to streptavidin M280 Dynabeads at ~500 ng DNA/10 μl bead slurry. 200 bp ultramers with Widom 601 nucleosome positioning sequence (table S1) were purchased from Integrated DNA Technologies and conjugated to streptavidin M280 Dynabeads at ~1 μg DNA/5 μl bead slurry. After conjugation, DNA-streptavidin beads were collected and incubated in 50 mM Tris-Cl, 0.25 mM EDTA, 0.05% Triton X-100 with 1 mM biotin for at least 30 min. DNA beads were extensively washed in sperm dilution buffer (5 mM HEPES, 100 mM KCl, 150 mM sucrose, 1 mM MgCl2, pH 8.0) prior to performing any pull-down assay. 5 μl DNA conjugated beads were incubated in 30 μl of interphase *Xenopus* egg extract, supplemented with 250 ng/μl cycloheximide and 300 μM aphidicolin. After incubation, beads were washed three times in bead wash buffer (10 mM K-HEPES, 50 mM Sucrose, 1 mM MgCl2, 100 mM KCl, 0.5 mM TCEP, 0.1% Triton-X). Beads were resuspended in 1x Laemmli buffer, boiled and supernatants were resolved by SDS-PAGE. Western blotting was performed against the indicated proteins. The loading order of the gel for each experimental repeat is indicated below. 20240206_exp1: Lane 1: Protein ladder Lane 2: Input (extract) Lane 3: pull down, no DNA Lane 4: pull down, non-Me Lane 5: pull down, full-Me Lane 6: pull down, hemi-Me Fw Lane 7: pull down, hemi-Me Rv Lane 8: blank Lane 9: Ladder Lane 10: 54bp DNA marker 20240214_exp2: Lane 1: Protein ladder Lane 2: Input (extract) Lane 3: pull down, no DNA Lane 4: pull down, non-Me Lane 5: pull down, full-Me Lane 6: pull down, hemi-Me Fw Lane 7: pull down, hemi-Me Rv Lane 8: blank Lane 9: blank Lane 10: Protein ladder 2040313_exp3: Lane 1: Protein ladder Lane 2: Input (extract) Lane 3: pull down, no DNA Lane 4: pull down, non-Me Lane 5: pull down, full-Me Lane 6: pull down, hemi-Me Fw Lane 7: pull down, hemi-Me Rv Lane 8: blank Lane 9: blank Lane 10: Protein ladder Includes an Excel file (Fig_1A_quantification. xlsx) summarizing the quantification of the data from the raw .tif files, used to make the graphs Fig 1B and 1C. Quantification of pulled-down CDCA7 signal relative to the DNA (SYBR Safe) signal are calculated. Description of 'Fig_1A_quantification' Excel sheet: Under the 'quantification' tab, the calculations are set up as follows: *20240214*: refers to the experiment number *un-Me*: refers to non-methylated DNA bead pull-down *full-Me*: refers to fully-methylated DNA bead pull-down *hemi-Me_Fw: refers to hemi-methylated DNA bead pull-down, where the methylated cytosine is placed in the forward strand *hemi-Me_Rv: refers to hemi-methylated DNA bead pull-down, where the methylated cytosine is placed in the reverse strand *HELLS*: signal of HELLS in each respective DNA pull-down *CDCA7*: signal of CDCA7 in each respective DNA pull-down *DNA*: signal of DNA (SYBR Safe) in each respective DNA pull-down *CDCA7/DNA*: CDCA7 divided by DNA *HELLS/DNA*: HELLS divided by DNA **Supporting files for Fig. 1D** Fig_1D.zip includes the raw tif files of the DNA bead pull-down experiment shown in Fig 1D. 35S-labeled *X. laevis*CDCA7e proteins (wildtype or with the indicated ICF3-patient associated mutation) were incubated with control beads, or beads conjugated 200 bp unmethylated or hemimethylated DNA (table S1). 35S-labeled xKid, a nonspecific DNA-binding protein, was used as a loading control. Autoradiography of 35S-labeled proteins in input and beads fraction is shown. To assess the in vitro binding of CDCA7e ICF mutants to hemimethylated DNA by autoradiography, 5 µl DNA beads were incubated in 20 µl binding buffer (10 mM HEPES, 100 mM NaCl, 0.025 % Triton X-100, 0.25 mM TCEP, pH 7.8) supplemented with 4 μl 35S-labeled CDCA7 and 1 μl 35S-labeled xKid. The loading order of the gel for each experimental repeat is indicated below. Lane 1: ladder Lane 2: In vitro-translated WT xCDCA7e Lane 3: In vitro-translated R232H xCDCA7e Lane 4: In vitro-translated G252V xCDCA7e Lane 5: In vitro-translated R262H xCDCA7e Lane 6: In vitro-translated xKid DBD Lane 7: Input; WT Lane 8: Input; R232H Lane 9: Input; G252V Lane 10:Input; R262H Lane 11: beads pull-down; no DNA; WT Lane 12: beads pull-down; no DNA; R232H Lane 13: beads pull-down; no DNA; G252V Lane 14: beads pull-down; no DNA; R262H Lane 15: beads pull-down; un-Me; WT Lane 16: beads pull-down; un-Me; R232H Lane 17: beads pull-down; un-Me; G252V Lane 18: beads pull-down; un-Me; R262H Lane 19: beads pull-down; hemi-Me; WT Lane 20: beads pull-down; hemi-Me; R232H Lane 21: beads pull-down; hemi-Me; G252V Lane 22: beads pull-down; hemi-Me; R262H Lane 23: ladder **Supporting files for Fig. 1E** Fig_1E.zip includes the raw tif file of the SDS-PAGE shown in Fig 1E. Recombinant XCDCA7e protein (WT and R232H) tagged with 3XFLAG at the N-terminus (3×FLAG-xCDCA7e) was expressed in Sf9 cells and purified with FLAG-M2 agarose. Both samples were resolved by SDS-PAGE, and visualized by Coomassie Blue staining. The loading order of the gel is indicated below. Lane 1: ladder Lane 2: purified 3XFLAG-xCDCA7e WT (1.0 μl) Lane 3: purified 3XFLAG-xCDCA7e R232H(1.0 μl) Lane 4: blank Lane 5: purified 3XFLAG-xCDCA7e WT(0.5 μl) Lane 6: purified 3XFLAG-xCDCA7e R232H(0.5 μl) Lane 7: blank Lane 8: ladder Lane 2: purified 3XFLAG-xCDCA7e WT(0.25 μl) Lane 3: purified 3XFLAG-xCDCA7e R232H(0.25 μl) **Supporting files for Fig. 1F** Fig_1F.zip includes the raw tif file of the EMSA shown in Fig 1F. 10 μl of samples (3XFLAG-xCDCA7e) were incubated for 30 min at 4 ˚C in a binding buffer [20 mM Tris-HCl (pH7.5) containing 150 mM NaCl, 1 mM DTT, 0.05 % NP-40 and 10% (w/v) glycerol] and electrophoresis was performed using a 0.5 × Tris-Acetate buffer [20 mM Tris-Acetic acid containing 0.5 mM EDTA at constant current of 8 mA for 100 min in a cold room on a 7.5% polyacrylamide gel purchased from Wako (SuperSepTM, Wako). 0.77, 1.54 and 3.85 equimolar excess of 3×FLAG-xCDCA7WT were added to the sample solution including 0.5 μM hemi-, full- and un-methylated DNA (upper: 5’- CAGGCAATCXGGTAGATC, lower: 5’-GATCTACXGGATTGCCTG, where X indicates cytosine or 5-methylcytosine, GeneDesign, Inc.) and the hemimethylated DNA substrate contains 5-methylcytosine in the upper strand, GeneDesign, Inc.). **Supporting files for Fig. 1G** Fig_1G.zip includes the raw tif file of the EMSA shown in Fig 1G. 10 μl of samples ( (3XFLAG-xCDCA7e and (3XFLAG-xCDCA7e R232H ) were incubated for 30 min at 4 ˚C in a binding buffer [20 mM Tris-HCl (pH7.5) containing 150 mM NaCl, 1 mM DTT, 0.05 % NP-40 and 10% (w/v) glycerol] and electrophoresis was performed using a 0.5 × Tris-Acetate buffer [20 mM Tris-Acetic acid containing 0.5 mM EDTA at constant current of 8 mA for 100 min in a cold room on a 7.5% polyacrylamide gel purchased from Wako (SuperSepTM, Wako). 0.77, 1.54 and 3.85 equimolar excess of 3×FLAG-xCDCA7 were added to the sample solution including 0.5 μM hemi-, full- and un-methylated DNA (upper: 5’- CAGGCAATCXGGTAGATC, lower: 5’-GATCTACXGGATTGCCTG, where X indicates cytosine or 5-methylcytosine, GeneDesign, Inc.) and the hemimethylated DNA substrate contains 5-methylcytosine in the upper strand, GeneDesign, Inc.). **Supporting files for Fig. 2B** Fig_2B.zip includes the raw tif file of the EMSA experiment shown in Fig 2B. 10 μl of samples ( (human CDCA7 264-371 ) were incubated for 30 min at 4 ˚C in a binding buffer [20 mM Tris-HCl (pH7.5) containing 150 mM NaCl, 1 mM DTT, 0.05 % NP-40 and 10% (w/v) glycerol] and electrophoresis was performed using a 0.5 × Tris-Acetate buffer [20 mM Tris-Acetic acid containing 0.5 mM EDTA at constant current of 8 mA for 100 min in a cold room on a 7.5% polyacrylamide gel purchased from Wako (SuperSepTM, Wako). 0.5, 1.0 and 2.0 equimolar excess of the CDCA7 264-371 were added to the sample solution including 0.5 μM hemi-, full- and un-methylated DNA (upper: 5’- CAGGCAATCXGGTAGATC, lower: 5’-GATCTACXGGATTGCCTG, where X indicates cytosine or 5-methylcytosine, GeneDesign, Inc.) and the hemimethylated DNA substrate contains 5-methylcytosine in the upper strand, GeneDesign, Inc.). **Supporting files for Fig. 2C-F** Includes the raw tif files for the DNA bead pull-down experiments shown in Fig. 2C-F. Magnetic beads coupled with double-stranded 54 bp DNA oligos containing unmethylated (un-Me), hemimethylated (hemi-me) or fully-methylated (full-Me) CpGs were incubated for 10 min in the presence of the indicated 35S-labeled CDCA7 homolog and 35S-labeled xKid proteins. SDS-PAGE gels were stained with SYBR-Safe to visualize loading of the 54 bp DNA. Representative autoradiographs of 35S-labeled proteins in input and DNA pull-downs are shown. For each experiment a scan of the DNA-stained gel (SYBR Safe) and Autoradiograph [Phosphor] are included. To assess binding of CDCA7 homologs, DNA bead pull-down was performed using 10 µl DNA conjugated beads incubated in 30 μl of interphase *Xenopus *egg extract (Fig. 2C) or boiled and clarified extract supernatant (Fig. 2D-F), supplemented with 3 μl 35S-labeled CDCA7 and 0.9 μl 35S-labeled xKid. Beads were washed and recovered three times, boiled in 1x Laemmli buffer, and supernatants were resolved by SDS-PAGE. For DNA quantitation, SDS-PAGE gel was stained with 0.01% SYBR-Safe (Thermo Fisher Scientific) for 15 minutes before imaging. Gel was fixed in fixative (1:2:7 glacial acetic acid:methanol: H2O), dried and exposed on a PhosphorImager screen. 54 bp DNA substrates (table S1) were conjugated to streptavidin M280 Dynabeads at ~500 ng DNA/10 μl bead slurry. 200 bp ultramers with Widom 601 nucleosome positioning sequence (table S1)* *were purchased from Integrated DNA Technologies and conjugated to streptavidin M280 Dynabeads at ~1 μg DNA/5 μl bead slurry. After conjugation, DNA-streptavidin beads were collected and incubated in 50 mM Tris-Cl, 0.25 mM EDTA, 0.05% Triton X-100 with 1 mM biotin for at least 30 min. DNA beads were extensive washed in sperm dilution buffer (5 mM HEPES, 100 mM KCl, 150 mM sucrose, 1 mM MgCl2, pH 8.0) prior to performing any pull-down assay. *In vitro *DNA pull-downs were performed in boiled and clarified extract supernatant where indicated (Fig. 2D-F) . Boiled and clarified egg extract supernatant was prepared by boiling CSF extract for 15 minutes followed by ultracentrifugation for 30 min at 260,000× g. Supernatant was aliquoted, frozen in liquid nitrogen and stored at -80 °C. All DNA pull-downs were performed at 20 °C. Loading order for each experiment is indicated below. 20240301_Arabidopsis: Lane 1: Ladder Lane 2: Input, A. thaliana Lane 3: no DNA, A. thaliana Lane 4: NM, A. thaliana Lane 5: HM, A. thaliana Lane 6: FM, A. thaliana 20240303_Xenopus_Arabidopsis: This data was used for representative figure Fig 2F Lane 1: Ladder Lane 2: Input, X. laevis Lane 3: no DNA, X. laevis Lane 4: NM, X. laevis Lane 5: HM, X. laevis Lane 6: FM, X. laevis Lane 7: blank lane Lane 8: blank lane Lane 9: Input, A. thaliana Lane 10: no DNA, A. thaliana Lane 11: NM, A. thaliana Lane 12: HM, A. thaliana Lane 13: FM, A. thaliana Lane 14: blank lane Lane 15: DNA marker 54bp 20240303_Nematostella_Gigas: Lane 1: Ladder Lane 2: Input, N. vectensis Lane 3: pull down; no DNA, N. vectensis Lane 4: pull down; non-Me, N. vectensis Lane 5: pull down; hemi-Me, N. vectensis Lane 6: pull down; full-Me, N. vectensis Lane 7: blank lane Lane 8: blank lane Lane 9: Input, C. gigas Lane 10: pull down; no DNA, C. gigas Lane 11: pull down; non-Me, C. gigas Lane 12: pull-down; hemi-Me, C. gigas Lane 13: pull-down; full-Me, C. gigas Lane 14: blank lane Lane 15: DNA marker 54bp 20240304_Nematostella_Gigas: Lane 1: Ladder Lane 2: Input, X. laevis Lane 3: pull-down; no DNA, X. laevis Lane 4: pull-down; non-Me, X. laevis Lane 5: pull-down; hemi-Me, X. laevis Lane 6: pull-down; full-Me, X. laevis Lane 7: blank lane Lane 8: Input, N. vectensis Lane 9: pull-down; no DNA, N. vectensis Lane 10: pull-down; non-Me, N. vectensis Lane 11: pull-down; hemi-Me, N. vectensis Lane 12: pull-down; full-Me, N. vectensis Lane 13: blank lane Lane 14: Input, C. gigas Lane 15: pull-down; no DNA, C. gigas Lane 16: pull-down; non-Me, C. gigas Lane 17: pull-down; hemi-Me, C. gigas Lane 18: pull-down; full-Me, C. gigas Lane 19: blank lane Lane 20: DNA marker 54bp 20240305_Xenopus_Arabidopsis_Nematostella: This data was used for the representative figure Fig 2D Lane 1: Ladder Lane 2: Input, X. laevis Lane 3: pull-down; no DNA, X. laevis Lane 4: pull-down; non-Me, X. laevis Lane 5: pull-down; hemi-Me, X. laevis Lane 6: pull-down; full-Me, X. laevis Lane 7: blank lane Lane 8: Input, A. thaliana Lane 9: pull-down; no DNA, A. thaliana Lane 10: pull-down; non-Me, A. thaliana Lane 11: pull-down; hemi-Me, A. thaliana Lane 12: pull-down; full-Me, A. thaliana Lane 13: blank lane Lane 14: Input, N. vectensis Lane 15: pull-down; no DNA, N. vectensis Lane 16: pull-down; non-Me, N. vectensis Lane 17: pull-down; hemi-Me, N. vectensis Lane 18: pull-down; full-Me, N. vectensis Lane 19: blank lane Lane 20: DNA marker 54bp 20240312_Xenopus_Arabidopsis_Nematostella: Lane 1: Ladder Lane 2: Input, X. laevis Lane 3: pull-down; no DNA, X. laevis Lane 4: pull-down; non-Me, X. laevis Lane 5: pull-down; hemi-Me, X. laevis Lane 6: pull-down; full-Me, X. laevis Lane 7: blank lane Lane 8: Input, A. thaliana Lane 9: pull-down; no DNA, A. thaliana Lane 10: pull-down; non-Me, A. thaliana Lane 11: pull-down; hemi-Me, A. thaliana Lane 12: pull-down; full-Me, A. thaliana Lane 13: blank lane Lane 14: Input, N. vectensis Lane 15: pull-down; no DNA, N. vectensis Lane 16: pull-down; non-Me, N. vectensis Lane 17: pull-down; hemi-Me, N. vectensis Lane 18: pull-down; full-Me, N. vectensis 20240313_hCDCA7_hCDCA7L: Lane 1: Ladder Lane 2: Input, hCDCA7 minimal domain Lane 3: pull-down; no DNA, hCDCA7 minimal domain Lane 4: pull-down; non-Me, hCDCA7 minimal domain Lane 5: pull-down; hemi-Me, hCDCA7 minimal domain Lane 6: pull-down; full-Me, hCDCA7 minimal domain Lane 7: blank lane Lane 8: Input, hCDCA7L minimal domain Lane 9: no DNA, hCDCA7L minimal domain Lane 10: pull-down; non-Me, hCDCA7L minimal domain Lane 11: pull-down; hemi-Me, hCDCA7L minimal domain Lane 12: pull-down; full-Me, hCDCA7L minimal domain Lane 13: blank lane Lane 14: DNA marker 54bp 20240314_Xenopus_Gigas: Lane 1: Ladder Lane 2: Input, X. laevis Lane 3: pull-down; no DNA, X. laevis Lane 4: pull-down; non-Me, X. laevis Lane 5: pull-down; hemi-Me, X. laevis Lane 6: pull-down; full-Me, X. laevis Lane 7: blank lane Lane 8: Input, C. gigas Lane 9: pull-down; no DNA, C. gigas Lane 10: pull-down; non-Me, C. gigas Lane 11: pull-down; hemi-Me, C. gigas Lane 12: pull-down; full-Me, C. gigas 20240321_hCDCA7L: Lane 1: Ladder Lane 2: Input, hCDCA7L minimal domain Lane 3: pull-down; no DNA, hCDCA7L minimal domain Lane 4: pull-down; non-Me, hCDCA7L minimal domain Lane 5: pull-down; hemi-Me, hCDCA7L minimal domain Lane 6: pull-down; full-Me, hCDCA7L minimal domain Lane 7: blank lane Lane 8: Input, hCDCA7L minimal domain Lane 9: pull-down; no DNA, hCDCA7L minimal domain Lane 10: pull-down; non-Me, hCDCA7L minimal domain Lane 11: pull-down; hemi-Me, hCDCA7L minimal domain Lane 12: pull-down; full-Me, hCDCA7L minimal domain Includes an Excel file (Fig_2C-F_quantification.xlsx) summarizing the quantification of the data from the raw tif files. Quantification of pulled-down 35S CDCA7 signal relative to the DNA signal are calculated. Description of 'Fig_2C-F_quantification' Excel sheet: Under the 'quantification' tab, the calculations are set up as follows: *AtCDCA7*: refers to CDCA7 homolog of Arabidopsis thaliana *NvCDCA7*: refers to CDCA7 homolog of Nematostella vectensis *CgCDCA7*: refers to CDCA7 homolog of Crassostrea Gigas *hCDCA7L*: refers to CDCA7 HMZF domain of the CDCA7L paralog in humans (aa 322-454 of NP_061189) *20240301*: refers to date of experiment *NM*: refers to non-methylated DNA bead pull-down *HM*: refers to non-methylated DNA bead pull-down *FM*: refers to non-methylated DNA bead pull-down *C7 Input*: signal of CDCA7 homolog in the Input *CDCA7*: signal of CDCA7 in each respective DNA pull-down *C7/Input*: signal of CDCA7 in each respective DNA pull-down normalized to the signal of CDCA7 in the Input *SYBR Safe*: signal of DNA (SYBR Safe) in each respective DNA pull-down *SYBR/sum*: signal of DNA (SYBR Safe) in each respective DNA pull-down normalized to the sum of SYBR Safe signals in each DNA pull-down *C7/Input:SYBR/sum*: normalized CDCA7 signal divided by normalized DNA (SYBR Safe) signal Under the 'Final data for graphs' tab, the numbers used to make the graphs in Figure 2C-F are summarized: *Organism*: refers to the organism from in which the CDCA7 homolog is found *Repeat*: indicates the experiment number *NM*: refers to non-methylated DNA bead pull-down *HM*: refers to non-methylated DNA bead pull-down *FM*: refers to non-methylated DNA bead pull-down *A. thaliana*: refers to Arabidopsis thaliana *N. vectensis*: refers to Nematostella vectensis *C. gigas*: refers to Crassostrea Gigas *hCDCA7L (HMZF domain): refers to CDCA7 HMZF domain of the CDCA7L paralog in humans (aa 322-454 of NP_061189) **Supporting files for Fig. 3A** Fig 3A.zip includes the raw .png file of the EMSA experiment shown in Fig 3A. 0.5, 1.0, 2.0 and 3.0 equimolar excess of the CDCA7 264-371 C339S were added to 0.1 µM nucleosomes in 10 µl reaction solution (binding buffer: 20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM DTT, 10 % Glycerol, 0.05 % NP-40) and electrophoresis was performed using a 0.5 × TBE buffer [(45 mM Tris-borate and 1 mM EDTA ) at constant current of 10 mA for 95 min in a cold room on a 7.5% polyacrylamide gel. To analyze the interactions, DNA was detected and analyzed by staining with GelRedTM (Wako) and the ChemiDoc XRS system (BIORAD), respectively. Raw_230927_EMSA_CDCA7C339S_NCP.png **Supporting files for Fig. 4A and B** Fig 4A-B.zip includes the raw tif file of all three EMSA experiment repeats shown in Fig 4A. Includes an Excel file summarizing the quantification of the data in Fig 4B: Fig_4B_quantification.xlsx 0.5, 1.0, 2.0 and 3.0 equimolar excess of the CDCA7 264-371 C339S were added to 0.1 µM nucleosomes in 10 µl reaction solution (binding buffer: 20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM DTT, 10 % Glycerol, 0.05 % NP-40) and electrophoresis was performed using a 0.5 × TBE buffer [(45 mM Tris-borate and 1 mM EDTA ) at constant current of 10 mA for 95 min in a cold room on a 7.5% polyacrylamide gel. To analyze the interactions, DNA was detected and analyzed by staining with GelRedTM (Wako) and the ChemiDoc XRS system (BIORAD), respectively. *NCP_Nuc-58W*: refers the DNA signal of nucleosome core particle with hemimethylated CpG sites positioned at location -58W, as described in paper *NCP_Nuc-58C*: refers the DNA signal nucleosome core particle with hemimethylated CpG sites positioned at location -58C, as described in paper *DNA_Nuc-58W*: refers the naked DNA signal with hemimethylated CpG sites positioned at location -58W, as described in paper *DNA_Nuc-58C*: refers the naked DNA signal particle with hemimethylated CpG sites positioned at location -58C, as described in paper *molar ratio of*: refers to the equimolar excess of CDCA7 added to 0.1 uM nucleosomes in the reaction *data1*: refers to the experimental repeat *%*: refers to the DNA signal of unbound DNA detected relative to the condition where no CDCA7 is added to the reaction *ave*: calculates the average of the normalized nucleosome or DNA signal across the three experimental repeats **Supporting files for Fig. 5F and G** Fig 5 F-G.zip includes the raw tif files of the IP experiments shown in Fig 5F and G respectively. Anti-HELLS and anti-CDCA7e antibodies (25 μg) were coupled to 100 μl Protein A Dynabeads for 1 h at RT and crosslinked with PierceTM BS3 (Thermo Fisher Scientific), following the manufacturer’s protocol. Antibody beads were washed extensively in sperm dilution buffer (5 mM HEPES, 100 mM KCl, 150 mM sucrose, 1 mM MgCl2, pH 8.0). UHRF1, HELLS and CDCA7 mutants were cloned into pCS2 vector by Gibson assembly and radiolabeled with EasyTagTM L-[35S]-Methionine (Perkin Elmer) using the TnT Coupled Reticulocyte Lysate System (Promega) according to the manufacturer’s instructions. Immunoprecipitation was performed in interphase egg extracts (Fig. 5F-G) supplemented with 250 ng/μl cycloheximide and the following ratios of Tnt lysates: 7 μl of the indicated 35S-labeled HELLS mutants with 1 μl 35S-labeled xCDCA7e in 50 μl of extract (Fig. 5F); 7 μl of the indicated 35S-labeled CDCA7 mutants with 1 μl 35S-labeled HELLS in 50 μl of extract (Fig. 5G). Extract was added to the beads and incubated on ice for 1 h with flicking every 20 min. The extract was diluted with 10 volumes CSF-XB (100 mM KCl, 1 mM MgCl2, 50 mM sucrose, 5 mM EGTA, and 10 mM HEPES, pH 8.0) and beads were recovered on a magnet. Beads were washed and recovered three times with 150 μl CSF-XB with 0.1 % Triton X-100. Beads were resuspended in 1x Laemmli buffer, boiled and supernatants were resolved by SDS-PAGE. Gels were fixed in fixative (1:2:7 glacial acetic acid:methanol:H2O), dried and exposed on a PhosphorImager screen. Control immunoprecipitation was performed using purified preimmune rabbit IgG (Sigma-Aldrich). **Supporting files for Fig. 6A and D** Fig 6A-D.zip includes the raw tif files of all three replicates of the DNA bead pull-down experiment shown in Fig 6A and the DNA bead pull-down shown in Fig 6D. **(A)** Beads coated with unmethylated (un-Me), hemimethylated (hemi-Me) or fully methylated (full-Me) 3 kb DNA (pBluescript) were incubated with interphase *Xenopus* mock IgG-depleted extract (∆MOCK), CDCA7e-depleted extract (∆CDCA7e) or HELLS-depleted extract (∆HELLS) for 10 min. Beads were isolated and western blotting analysis was perfomed. **(D)** 35S-labeled HELLS or HELLS ∆63-96 was incubated with beads coated with 200 bp unmethylated or hemimethylated DNA for 30 min in interphase *Xenopus* egg IgG-depleted or CDCA7-depleted extracts. Beads were isolated and associated 35S-labeled proteins were visualized by autoradiography. Includes an Excel file (Fig_6A_quantification.xlsx) summarizing the quantification of the data in Fig 6A. Quantification of pulled-down HELLS or CDCA7 signal relative to the H3 signal are calculated. Description of 'Fig_6A_quantification' Excel sheet: Under the 'Raw data quantification' tab, the calculations are set up as follows: *20240207*: indicates the experimental repeat *un-Me*: refers to non-methylated DNA bead pull-down *hemi-me*: refers to hemi-methylated DNA bead pull-down *fully-me*: refers to fully-methylated DNA bead pull-down *mock-depleted*: refers to mock-depleted extract *CDCA7-depleted*: refers to CDCA7-depleted extract *HELLS-depleted*: refers to HELLS-depleted extract *H3*: H3 signal detected for DNA pull-down *CDCA7*: CDCA7 signal detected for DNA pull-down *HELLS*: HELLS signal detected for DNA pull-down *CDCA7/H3*: CDCA7 signal normalized to H3 signal *HELLS/H3*: HELLS signal normalized to H3 signal Under the 'Final data for graphs' tab, the numbers used to make the graphs in Figure 6B and C are summarized: *Figure 6B*: refers to the figure where graph is found *un-Me*: refers to non-methylated DNA bead pull-down *hemi-me*: refers to hemi-methylated DNA bead pull-down *fully-me*: refers to fully-methylated DNA bead pull-down *Δmock*: refers to mock-depleted extract *ΔCDCA7*: refers to CDCA7-depleted extract *ΔHELLS*: refers to HELLS-depleted extract To generate biotinylated hemimethylated pBlueScript DNA substrates (for Fig. 6A), a PCR-linearized pBlueScript template was methylated by the CpG methyltransferase M.SssI according to manufacturer’s protocol (Cat #EM0821, Thermo Fisher Scientific). DNA synthesis across the methylated linearized pBlueScript template was subsequently performed in Q5® High-Fidelity 2X Master Mix (New England Biolabs, Inc.) using a 5’ biotinylated primer (5’-/5Biosg/CGTTCTTCGGGGCGAAAACTCTCAAGG -3’) purchased from Integrated DNA Technologies. The reaction mix was purified using the QIAquick PCR purification kit (QIAGEN) and the resultant hemimethylated DNA product was subsequently purified from the reaction mix by conjugation to streptavidin M280 Dynabeads (Invitrogen). For nonmethylated 3 kb DNA substrates, the above protocol was performed using unmethylated linearized pBlueScript DNA template during DNA synthesis. Fully-methylated pBlueScript DNA substrates were generated by methylating the nonmethylated pBlueScript DNA substrates with CpG methyltransferase M.SssI (Thermo Fisher Scientific) prior to DNA-bead conjugation. Methylation status of all BlueScript DNA substrates was confirmed by restriction digest with BstUI (New England Biolabs, Inc.). BlueScript DNA substrates were coupled to streptavidin beads at ~2 μg DNA/5 μl bead slurry in bead coupling buffer (50 mM Tris-Cl, 0.25 mM EDTA, 0.05% Triton X-100, pH 8.0) supplemented with 2.5% polyvinyl alcohol and 1.5 M NaCl for at least 2 h at RT. 200 bp ultramers with Widom 601 nucleosome positioning sequence (Fig. 6D, table S1) were purchased from Integrated DNA Technologies and conjugated to streptavidin M280 Dynabeads at ~1 μg DNA/5 μl bead slurry. After conjugation, DNA-streptavidin beads were collected and incubated in 50 mM Tris-Cl, 0.25 mM EDTA, 0.05% Triton X-100 with 1 mM biotin for at least 30 min. DNA beads were extensive washed in sperm dilution buffer (5 mM HEPES, 100 mM KCl, 150 mM sucrose, 1 mM MgCl2, pH 8.0) prior to performing any pull-down assay. As boiled supernatants of cell lysates are known to prevent proteins from binding to beads nonspecifically (*68*), *in vitro *DNA pull-downs were performed in boiled and clarified extract supernatant where indicated (Fig. 2D-F) (*68*). Boiled and clarified egg extract supernatant was prepared by boiling CSF extract for 15 minutes followed by ultracentrifugation for 30 min at 260,000× g. Supernatant was aliquoted, frozen in liquid nitrogen and stored at -80 °C. All DNA pull-downs were performed at 20 °C. For DNA bead pull-downs analyzed by western blot (Fig. 6A) 5 μl DNA conjugated beads were incubated in 30 μl of interphase *Xenopus* egg extract, supplemented with 250 ng/μl cycloheximide and 300 μM aphidicolin. After incubation, beads were washed three times in bead wash buffer (10 mM K-HEPES, 50 mM Sucrose, 1 mM MgCl2, 100 mM KCl, 0.5 mM TCEP, 0.1% Triton-X). Beads were resuspended in 1x Laemmli buffer, boiled and supernatants were resolved by SDS-PAGE. Western blotting was performed against the indicated proteins. To assess the recruitment of HELLS to hemimethylated DNA in egg extract using autoradiography (Fig. 6D), 5 μl DNA conjugated beads were incubated in 30 µl interphase *Xenopus* egg extract supplemented with 250 ng/μl cycloheximide, 300 μM aphidicolin 3μl 35S-labeled HELLS and 0.9 μl 35S-labeled xKid-DBD per μl of extract. Beads were washed and recovered three times, boiled in 1x Laemmli buffer, and supernatants were resolved by SDS-PAGE. For DNA quantitation, SDS-PAGE gel was stained with 0.01% SYBR-Safe (Thermo Fisher Scientific) for 15 minutes before imaging. Gel was fixed in fixative (1:2:7 glacial acetic acid:methanol: H2O), dried and exposed on a PhosphorImager screen. **Supporting files for Fig. 7A-C** Figure 7A.zip includes the raw tif files of the western blot shown in Figure 7A probed with the indicated antibody. Figure 7B.zip includes the raw tif files of the western blot shown in Figure 7B probed with the indicated antibody. Figure 7C.zip includes the raw tif files of all three experimental repeats for the experiment shown in Figure 7B. Includes an Excel file (Fig_7C_quantification.xlsx) summarizing the quantification of the data.   Fig. 7A. *Xenopus* sperm nuclei were incubated for 120 min in interphase *Xenopus* egg extract in the presence of 1.1 μM recombinant mDPPA3. Chromatin was isolated and reincubated in interphase egg extract in the presence or absence of 150 μM aphidicolin (APH).* *Chromatin was then isolated at 20, 40 and 60 min after transfer and chromatin-bound proteins were analyzed by western blotting using indicated antibodies. The loading order of the gel is indicated below. Lane 1: Ladder Lane 2: egg extract (+DMSO) Lane 3: egg extract (+APH) Lane 4: blank Lane 5: chromatin; +DMSO, 0 min Lane 6: chromatin; +DMSO, 20 min Lane 7: chromatin; +DMSO, 40 min Lane 8: chromatin; +DMSO, 60 min Lane 9: chromatin; +APH, 20 min Lane 10: chromatin; +APH, 40 min Lane 11: chromatin; +APH, 60 min Fig. 7B. Sperm nuclei were incubated for 120 min in Mock-depleted extracts or either CDCA7e-depleted or HELLS-depleted extracts supplemented with mDPPA3. Chromatin was isolated and reincubated in Mock-depleted and either CDCA7e-depleted or HELLS-depleted extracts in the presence of aphidicolin. Chromatin was then isolated at 0 and 2 min and chromatin-bound proteins were analyzed by western blotting using indicated antibodies. The loading order of the gel is indicated below. Lane 1: Ladder Lane 2: egg extract (Mock-depleted) Lane 3: egg extract (CDCA7e-depleted) Lane 4: egg extract (HELLS-depleted) Lane 5: blank Lane 6: chromatin; Mock-depleted, 0 min Lane 7: chromatin; CDCA7e-depleted, 0 min Lane 8: chromatin; HELLS-depleted, 0 min Lane 9: chromatin; Mock-depleted, 2 min Lane 10: chromatin; CDCA7e-depleted, 2 min Lane 11: chromatin; HELLS-depleted, 2 min Lane 12: chromatin; Mock-depleted, 5 min Lane 13: chromatin; CDCA7e-depleted, 5 min Lane 14: chromatin; HELLS-depleted, 5 min Lane 15: chromatin; Mock-depleted, 15 min Lane 16: chromatin; CDCA7e-depleted,15 min Lane 17: chromatin; HELLS-depleted, 15 min   Fig_7C_quantification.xlsx summarizes the quantification of the data in Fig 7C. *Exp.1*: refers to experimental repeat *Relative amount*: refers to the normalized Western Blot signal at the 2 minute timepoint of the indicated protein normalized to the signal ORC2. This ratio is normalized to the signal in mock-depleted extract *Mock*: refers to mock-depleted extract *CDCA7*: refers to CDCA7-depleted extract *HELLS*: refers to HELLS-depleted extract **Supporting files for Fig. S2A** Fig S2A.zip includes the raw tif files of the western blot shown in Figure S2A. Also includes the contrast adjusted version of the file used to assemble the final figure shown in S2A. Magnetic beads coupled with 3 kb pBluescript DNA with unmethylated CpGs (un-Me), hemimethylated CpGs (hemi-Me), or fully methylated CpGs (full-Me), were incubated with interphase *Xenopus *egg extracts. Beads were collected after 60 min and analyzed by western blotting. **Supporting files for Fig. S2B** Fig S2B.zip includes the raw tif files of the western blot shown in Figure S2B probed with the indicated antibody. *Xenopus* sperm nuclei were isolated at indicated time points after incubation with interphase *Xenopus* egg extracts in the presence or absence of 0.5 μM mouse DPPA3 (mDPPA3). Chromatin-associated proteins were analyzed by western blotting. The loading order of the gel is indicated below. Lane 1: Ladder Lane 2: egg extract (+buffer) Lane 3: egg extract (+mDPPA3) Lane 4: egg extract (+mDPPA361-150) Lane 5: blank Lane 6: chromatin; +buffer, 20 min Lane 7: chromatin; +buffer, 40 min Lane 8: chromatin; +buffer, 60 min Lane 9: chromatin; +buffer, 80 min Lane 10: chromatin; +mDPPA3, 20 min Lane 11: chromatin; +mDPPA3, 40 min Lane 12: chromatin; +mDPPA3, 60 min Lane 13: chromatin; +mDPPA3, 80 min Lane 14: chromatin; +mDPPA361-150, 20 min Lane 15: chromatin; +mDPPA361-150, 40 min Lane 16: chromatin; +mDPPA361-150, 60 min Lane 17: chromatin; +mDPPA361-150, 80 min   **Supporting files for Fig. S2C** Fig S2C.zip includes the raw tif files of the western blot shown in Figure S2C probed with the indicated antibody. *Xenopus* sperm nuclei were isolated at indicated time points after incubation with interphase *Xenopus* egg extracts in the presence or absence of 0.5 μM recombinant geminin. At each time point, chromatin-associated proteins were analyzed by western blotting. The loading order of the gel is indicated below. Lane 1: Ladder Lane 2: egg extract (+buffer) Lane 3: egg extract (+geminin) Lane 4: blank Lane 5: chromatin; +buffer, 30 min Lane 6: chromatin; +buffer, 60 min Lane 7: chromatin; +buffer, 90 min Lane 8: chromatin; +buffer, 120 min Lane 9: chromatin; +geminin, 30 min Lane 10: chromatin; +geminin, 60 min Lane 11: chromatin; +geminin, 90 min Lane 12: chromatin; +geminin, 120 min **Supporting files for Fig. S2D** Fig S2D.zip includes the raw tif files of the autoradiograph shown in Figure S2D. Immunoprecipitation by control IgG or anti-HELLS antibody from *Xenopus *egg extracts containing 35S-labeled UHRF1 and 35S-labeled CDCA7e. Immunoprecipitation was performed in interphase egg extracts supplemented with 250 ng/μl cycloheximide and the following ratios of Tnt lysates: 2 μl 35S-labeled UHRF1 and 2 μl 35S-labeled CDCA7 per 25 μl of extract. Extract was added to the beads and incubated on ice for 1 h with flicking every 20 min. The extract was diluted with 10 volumes CSF-XB (100 mM KCl, 1 mM MgCl2, 50 mM sucrose, 5 mM EGTA, and 10 mM HEPES, pH 8.0) and beads were recovered on a magnet. Beads were washed and recovered three times with 150 μl CSF-XB with 0.1 % Triton X-100. Beads were resuspended in 1x Laemmli buffer, boiled and supernatants were resolved by SDS-PAGE. Gels were fixed in fixative (1:2:7 glacial acetic acid:methanol:H2O), dried and exposed on a PhosphorImager screen. Control immunoprecipitation was performed using purified preimmune rabbit IgG (Sigma-Aldrich). The gel loading order is indicated below: Lane 1: In vitro-translated UHRF1 Lane 2: Input (in-vitro translated URHF1 and CDCA7 in extract) Lane 3: IP IgG Lane 4: IP HELLS Lane 9: Ladder **Supporting files for Fig. S2E** Fig S2E.zip includes the raw tif files of the autoradiograph shown in Figure S2E. Immunoprecipitation by control IgG or anti-UHRF1 serum from *Xenopus *egg extracts containing 35S-labeled HELLS and 35S-labeled UHRF1. Immunoprecipitation was performed in interphase egg extracts (Fig. 5F-G) supplemented with 250 ng/μl cycloheximide and the following ratios of Tnt lysates: 3.5 μl 35S-labeled HELLS with 0.5 μl 35S-labeled UHRF1 per 25 μl of extract. Extract was added to the beads and incubated on ice for 1 h with flicking every 20 min. The extract was diluted with 10 volumes CSF-XB (100 mM KCl, 1 mM MgCl2, 50 mM sucrose, 5 mM EGTA, and 10 mM HEPES, pH 8.0) and beads were recovered on a magnet. Beads were washed and recovered three times with 150 μl CSF-XB with 0.1 % Triton X-100. Beads were resuspended in 1x Laemmli buffer, boiled and supernatants were resolved by SDS-PAGE. Gels were fixed in fixative (1:2:7 glacial acetic acid:methanol:H2O), dried and exposed on a PhosphorImager screen. Control immunoprecipitation was performed using purified preimmune rabbit IgG (Sigma-Aldrich). The gel loading order is indicated below: Lane 1: Input (in vitro-translated UHRF1 and HELLS in extract) Lane 2: IP IgG Lane 3: IP UHRF1 Lane 4: blank Lane 5: ladder **Supporting files for Fig. S3** Fig_S3.zip includes the raw tif file of the EMSA shown in Fig S3B-D (as described in the methods section of the paper) and the raw tif file of the SDS-PAGE shown in Fig S3E. (B-D) Native gel electrophoresis mobility shift assay for detecting the interaction of hCDCA7 264-340 (B) hCDCA7 235-340 (C), and hCDCA7 264-371 C339S (D) with double stranded DNA oligonucleotides with a hemi-methylated (hemi-Me), fully- methylated (full-Me) or unmethylated (un-Me) CpG. (E) Coomassie-stained gel of the indicated purified hCDCA7 fragments. 10 µl of samples were incubated for 30 min at 4 °C in a binding buffer [20 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 1 mM DTT, 0.05 % NP-40 and 10% (w/v) glycerol] and electrophoresis was performed using a 0.5 × Tris-Acetate buffer [20 mM Tris-Acetic acid containing 0.5 mM EDTA at constant current of 8 mA for 100 min in a cold room on a 7.5% polyacrylamide gel purchased from Wako (SuperSepTM, Wako). 0.5, 1.0 and 2.0 equimolar excess of the CDCA7 264-371 or 3.0, 5.0 and 10.0 equimolar excess of the CDCA7 264-340 and 235-340 were added to the sample solution including 0.5 µM hemi-, full- and un-methylated DNA (upper: 5’- CAGGCAATCXGGTAGATC, lower: 5’-GATCTACXGGATTGCCTG, where X indicates cytosine or 5-methylcytosine and the hemimethylated DNA substrate contains 5-methylcytosine in the upper strand, GeneDesign, Inc.). **Supporting files for Fig. S6** Fig_S6.zip includes the raw tif file for the repeated experiments of the EMSA shown in Fig S6A and S6D (as described in the methods section of the paper). (A) Native gel electrophoresis mobility shift assay analyzing the interaction of human UHRF1 SRA domain with nucleosomes carrying hemimethylated CpG at the indicated positions (Table S2). (D) Native gel electrophoresis mobility shift assay analyzing the interaction of human UHRF1 SRA domain with nucleosomes carrying hemimethylated CpG at the indicated positions (Table S2). 0.5, 1.0, 2.0 and 3.0 equimolar excess of the CDCA7 264-371 or SRA 399-680 were added to 0.1 µM nucleosomes in 10 µl reaction solution (binding buffer: 20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM DTT, 10 % Glycerol, 0.05 % NP-40) and electrophoresis was performed using a 0.5 × TBE buffer [(45 mM Tris-borate and 1 mM EDTA ) at constant current of 10 mA for 95 min in a cold room on a 7.5% polyacrylamide gel. To analyze the interactions, DNA was detected and analyzed by staining with GelRedTM (Wako) and the ChemiDoc XRS system (BIORAD), respectively. **Supporting files for Fig. S10** Fig_S10.zip includes the raw tif file for the repeated experiments of immunoprecipitation shown in Fig S10 (as described in the methods section of the paper). FLAG M2 agarose were pre-incubated with Sf9 lysates expressing Wildtype (WT) or truncated versions of recombinant FLAG×3-tagged *X. laevis* CDCA7e proteins. FLAG-M2 agarose were then incubated with *Xenopus* egg extracts. Isolated proteins were analyzed by western blotting using anti-HELLS and anti-FLAG antibodies. The loading order of the gel is indicated below. Lane 1: Ladder Lane 2: egg extract (+total) Lane 3: FLAG M2 beads pull-down (pre-incubated with control Sf9 lysates) Lane 4: FLAG M2 beads pull-down (pre-incubated with Sf9 lysates expressing xCDCA7e WT) Lane 5: FLAG M2 beads pull-down (pre-incubated with Sf9 lysates expressing xCDCA7e 1-204) Lane 6: FLAG M2 beads pull-down (pre-incubated with Sf9 lysates expressing xCDCA7e 102-332) Lane 7: FLAG M2 beads pull-down (pre-incubated with Sf9 lysates expressing xCDCA7e 1205-332) Lane 8: FLAG M2 beads pull-down (pre-incubated with Sf9 lysates expressing xCDCA7e  D74-101) Lane 9: blank Lane 10: In vitro translated FLAG×3-HELLS   **Supporting files for Fig. S11** Fig_S11_quantification.xlsx summarizes the raw data (CPM) used to generate the graphs shown in Fig. S11. *X. laevis *sperm nuclei (A) or erythrocyte nuclei (B) were incubated with egg extracts for 60 min with *S*-[methyl-3H]-adenosyl-L-methionine with or without geminin, which inhibits DNA replication initiation. Radioactivity associated with chromosomal DNA is measured. DNA methylation of replicating sperm or erythrocyte nuclei in egg extract was assayed by the incorporation of 3H-SAM (S-[methyl-3H]-adenosyl-L-methionine; Perkin Elmer, NET155H). Demembranated sperm nuclei were prepared. Erythrocyte nuclei were prepared from blood collected from dead adult male *Xenopus laevis* frogs that were sacrificed for testis dissection. Erythrocyte nuclei were stored at -20 °C in 50% glycerol STMN buffer (10 mM NaCl, 10 mM Tris pH 7.4, 3 mM MgCl2, 0.5% NP-40). Sperm or erythrocyte nuclei were replicated in cycling egg extract (3000 nuclei/μl extract) supplemented with 250 ng/μl cycloheximide and 0.335 μM 3H-SAM (82.3 Ci/mmol) for 1 h at 20 °C. Replication was inhibited by the addition of 200 nM of recombinant GST-tagged nondegradable geminin. The reaction was stopped by the addition of 9 volumes of CPB. Genomic DNA was purified using a Wizard Genomic DNA Purification Kit (Promega) according to the manufacturer’s instructions. DNA pellets were resuspended in scintillation fluid (ScintiVerse; Thermo Fisher Scientific) and quantified using a liquid scintillation counter (Perkin Elmer, Tri-Carb® 2910 TR). *Biological repeat*: refers to biological repeat associated with the data *Technical repeat*: refers to the technical repeat performed within each biological repeat *ΔIgG*: refers to mock-depleted extract *ΔIgG+GEM*: refers to mock-depleted extract with Geminin *ΔCDCA7*: refers to CDCA7-depleted extract *ΔCDCA7+GEM*: refers to CDCA7-depleted extract with Geminin *ΔHELLS*: refers to HELLS-depleted extract *ΔHELLS+GEM*: refers to HELLS-depleted extract with Geminin *sperm*: refers to the nuclei added to the extract being sperm *erythrocyte*: refers to the nuclei added to the extract being erythrocyte **Supporting files for Fig. S12** Fig_S12_quantification.xlsx summarizes the raw data (CPM) used to generate the graphs shown in Fig. S12. To monitor DNA replication in egg extracts, [α-32P] dATP (3,000 Ci/mmol, Perkin Elmer) and sperm nuclei were added to interphase extracts and incubated at 22˚C. At each time point, extracts were diluted in reaction stop solution (1% SDS, 40 mM EDTA) and treated with Proteinase K (NACALAI TESQUE, Inc.) at 37 ˚C. The solutions were spotted onto Whatman glass microfiber filters followed by 5% trichloroacetic acid (TCA) containing 2%pyrophosphate. Filters were washed twice in ethanol and dried. The incorporation of radioactivity was counted in the scintillation cocktail. *Time*: Refers to the time of sperm nuclei incubation in egg extract *Mock-depleted*: Refers to the extract being mock-depleted *CDCA7-depleted*: Refers to the extract being CDCA7-depleted