**Structure and function of the conserved core of histone deposition
protein Asf1**

**Sally M. Daganzo\*†‡, Jan P. Erzberger†, Wendy M. Lam\*†,**

**Emmanuel Skordalakes†, Rugang Zhang#, Alexa A. Franco\*†, Steven J.
Brill\*\*,**

**Peter D. Adams#, James M. Berger† and Paul D. Kaufman\*†§**

\*Lawrence Berkeley National Laboratory, and

†University of California, Berkeley

**Department of Molecular and Cell Biology**

**Berkeley, CA 94720**

#Fox Chase Cancer Center

Philadelphia, PA 19111

\*\*Rutgers University

Dept. of Molecular Biology and Biochemistry

CABM - 679 Hoes Lane

Piscataway, NJ 08854

‡Present address:

University of California, San Francisco

San Francisco, CA 94143

§ Corresponding Author:

351 Donner Laboratory

Berkeley, CA 94720

Phone (510) 486-5846

**FAX (510) 486-6488**

**[pdkaufman@lbl.gov]{.underline}**

**Running head: Structure of histone deposition protein Asf1**

**Summary**

**[Background:]{.underline} Asf1 is a ubiquitous eukaryotic
histone-binding and deposition protein that mediates nucleosome
formation *in vitro* and is required for genome stability *in vivo*.
Studies in a variety of organisms have defined Asf1's role as a histone
chaperone during DNA replication through specific interactions with
histones H3/H4 and the histone deposition factor CAF-I. In addition to
its role in replication, conserved interactions with proteins involved
in chromatin silencing, transcription, chromatin remodeling and DNA
repair have also established Asf1 as an important component of a number
of chromatin assembly and modulation complexes.**

**[Results:]{.underline} We demonstrate that the highly conserved
N-terminal domain of *S.cerevisiae* Asf1 (Asf1N) is the core region that
mediates all tested functions of the full-length protein. The crystal
structure of this core domain, determined to 1.5 Å resolution, reveals a
compact immunoglobulin-like β-sandwich fold topped by three helical
linkers. The surface of Asf1 displays a conserved hydrophobic groove
flanked on one side by an area of strong electronegative surface
potential. These regions represent potential binding sites for histones
and other interacting proteins. The structural model also allowed us to
interpret mutagenesis studies of the human Asf1a/HIRA interaction and to
functionally define the region of Asf1 responsible for Hir1-dependent
telomeric silencing in budding yeast.**

**[Conclusions:]{.underline} The evolutionarily conserved, N-terminal
155 amino acids of histone deposition protein Asf1 are functional *in
vitro* and *in vivo*. This core region of Asf1 adopts a compact
immunoglobulin-fold structure with distinct surface characteristics,
including a Hir protein binding region required for gene silencing.**

**Introduction**

The fundamental structural units of eukaryotic chromosomes are
nucleosomes, particles composed of DNA wrapped around octamers of
histone proteins. During replication, new nucleosomes are assembled
through the directed deposition of histones onto newly replicated DNA
\[1, 2\]. Nucleosome assembly is a two-step process initiated by the
deposition of a histone (H3**.**H4)2 tetramer on DNA, followed by the
addition of two histone (H2A**.**H2B) dimers. This ordered mechanism of
histone addition is critical for proper nucleosome formation *in vivo*,
and is regulated by nucleosome assembly factors that bind histones and
mediate their interactions with DNA (reviewed in \[3, 4\].

One nucleosome assembly protein, Asf1, was originally discovered through
genetic studies in budding yeast as a factor that antagonized
chromatin-mediated gene silencing when overexpressed \[5, 6\].
*S.cerevisiae* cells lacking Asf1 display increased frequencies of
spontaneous genome rearrangements \[7\], non-viability in the presence
of double strand DNA breaks \[8\], and a dependence on genes involved in
transcriptional elongation for viability \[9\]. The fission yeast
homolog of Asf1, the CIA1 protein, is essential for viability \[10\].

Several biochemical studies have directly linked Asf1 to chromatin
assembly following DNA replication. For example, *Drosophila* Asf1 binds
newly synthesized histone H3/H4 tetramers to form a complex that
stimulates the histone deposition activity of Chromatin Assembly
Factor-I (CAF-I), through a direct interaction with its Cac2/p60 subunit
\[11-14\]. Asf1 also interacts directly with chromatin assembly factors
of the Hir1/HIRA family to support the formation of silent chromatin and
to regulate histone gene transcription and S-phase progression
\[15-20\]. Asf1 may also mediate chromatin assembly following DNA damage
through a direct interaction with the S-phase damage checkpoint kinase
Rad53 \[21, 22\].

In addition to its function in nucleosome assembly during DNA
replication and DNA repair, Asf1 has conserved roles in other aspects of
DNA metabolism. Asf1 has been shown to interact with the histone
acetyltransferase complex SAS-I \[15, 23, 24\], the chromatin remodeling
enzyme Brahma \[25\], and the basal transcription factor TFIID component
TAFII250/CCG1 \[26, 27\]. The complex interplay of Asf1 with its various
partner proteins is illustrated by the fact that Asf1 binding to Rad53
and histones H3/H4 is mutually exclusive \[21, 22\]. Similarly, Asf1
occludes bound histones from modification by the SAS-I histone
acetyltransferase complex \[28\], suggesting that these proteins may
compete for a small number of binding surfaces. To date, there is little
understanding of the molecular details that enable Asf1 to mediate such
a diverse array of functions.

As a first step towards understanding Asf1 function, we report here
biochemical and structural studies of the evolutionarily conserved
N-terminal domain of the Asf1 protein. Our results show that this core
domain adopts a β-sandwich fold that is sufficient to recapitulate
various aspects of full-length Asf1 function. Structural features and
residue conservation reveal regions of the protein that are candidate
protein interaction surfaces, and have allowed us to define an Hir1
interaction surface on Asf1 responsible for *in vivo* telomere
silencing.

**Results and Discussion**

**Asf1 amino acids 1-155 (Asf1N) are functional *in vivo* and *in
vitro***

*Saccharomyces cerevisiae* Asf1 comprises 279 amino acids distributed
into two distinct regions. The first 155 amino acids are highly
conserved among eukaryotic species (65-90% identity) and have predicted
secondary structure elements. The C-terminal region (\<15% identity) is
poorly conserved and is predicted to consist largely of disordered
coils. The C-terminal region does contains long poly-acidic tracts in
fungi, but this feature is not conserved in other eukaryotes. For
example, in *Leishmania major* Asf1, the C-terminal region is lacking
altogether. These observations, together with a previous study showing
that a fragment of the *S. cerevisiae* *ASF1* gene comprising amino
acids 1-169 is sufficient to restore viability to fission yeast mutants
lacking the homologous *cia1* gene \[10\], suggested that a minimal core
region comprising the conserved N-terminal 155 amino acids of budding
yeast Asf1 might be biologically functional. We will refer to this
N-terminal region as the Asf1N protein.

*S.cerevisiae* cells lacking the *ASF1* gene are sensitive to the DNA
damaging agent methyl methanesulfonate (MMS) and to the ribonucleotide
reductase inhibitor hydroxyurea (HU) \[11\]. To determine whether Asf1N
could complement the DNA damage sensitivity of *asf1∆* strains, we
introduced plasmids expressing Asf1 or Asf1N and exposed the cells to
MMS or HU. Our results demonstrate that the N-terminal domain of Asf1 is
as proficient as the wild type protein to confer resistance to DNA
damaging agents *in vivo* (Figure 1A).

Asf1 stimulates the activity of Chromatin Assembly Factor-I (CAF-I) *in
vitro* and contributes to chromatin-mediated silencing *in vivo* in a
manner that overlaps CAF-I \[11-14, 16\]. Yeast cells lacking both the
*ASF1* gene and any of the three CAF-I subunits encoded by the *CAC1*,
*2* and *3* genes display slow growth and possess poor
chromatin-mediated gene silencing \[11, 16\]. To define the region of
Asf1 responsible for mediating these effects, we compared growth rates
and telomere-proximal gene silencing in *cac1*∆ *asf1*∆ double mutants
expressing either full-length Asf1 or the truncated Asf1N protein
(Figure 1B). In our growth assays, plasmids encoding either Asf1 or
Asf1N restored wild-type growth rates to *cac1*∆ *asf1*∆ cells (Figure
1B, left panel). To assess gene silencing, we used a *URA3* reporter
gene inserted adjacent to telomere VIIL and measured growth on media
containing FOA, a metabolic toxin that positively selects for the
ability to silence the *URA3* gene \[29\]. As previously observed,
*cac1*∆ mutants display a partial loss of telomeric silencing, and
*cac1*∆ *asf1*∆ double mutants display a severe loss of silencing \[11,
30\]. In the *cac1*∆ *asf1*∆ double mutant cells, however, plasmids
encoding either Asf1 or Asf1N restored telomeric silencing to levels
observed in *cac1*∆ cells (Figure 1B, right panel). Taken together,
these data provide strong support that Asf1 and Asf1N are equally
competent to mediate the assembly of silenced chromatin *in vivo*.

*In vitro*, Asf1 is a histone H3/H4 binding and deposition protein \[10,
11, 16, 31\]. To determine whether Asf1N retains this function *in
vitro*, we performed co-precipitation experiments with purified histones
H3/H4 and recombinant, epitope-tagged Asf1 and Asf1N. Both Asf1 and
Asf1N retained similar amounts of histones after extensive washing with
buffer containing 250 mM NaCl, demonstrating that Asf1N is a functional
histone binding protein (Figure 1C). Histone binding to Asf1N was more
easily disrupted by high salt concentrations, however, suggesting that
the acidic C-terminus interacts with the H3/H4 tetramer, perhaps through
electrostatic contacts with the basic histone proteins.

Another binding partner of Asf1 is the Rad53 protein \[21, 22\], a cell
cycle checkpoint kinase that interacts with Asf1 through one of its
forkhead-associated (FHA) domains \[32\]. In co-precipitation
experiments, Asf1N retained similar amounts of Rad53 compared to Asf1
(Figure 1D), demonstrating that Asf1N is a functional Rad53-binding
protein. These data are consistent with the ability of Asf1N to provide
protection from DNA damaging agents (Figure 1A). In addition, the
interaction between Rad53 and Asf1N depended on enzymatic
dephosphorylation of the Rad53 protein preparation (data not shown).
This finding agrees with previous data demonstrating that the Asf1-Rad53
interaction is regulated by phosphorylation of Rad53 \[21, 22, 32\].

*In vitro*, Asf1 stimulates histone deposition by substoichiometric
amounts of CAF-I \[11, 13\]. To test whether Asf1N could also stimulate
histone deposition by CAF-I, we turned to *in vitro* DNA replication
reactions. In these assays, nucleosome formation is detected by the
supercoiling of circular, replicated DNA templates that have been
labeled by incorporation of a radioactive deoxynucleotide. We observed
that both full-length Asf1 and Asf1N were able to stimulate nucleosome
formation by a substoichiometric amount of CAF-I (Figure 1E), and that
the specific activity of Asf1N in this assay was as high, if not higher,
than that of full-length Asf1. As observed previously for Asf1, the
amount of Asf1N required to stimulate CAF-I was unable to promote
efficient histone deposition alone (lanes 6-8 and 12-14; \[11, 13\]),
reflecting the functional synergy between these histone deposition
factors. We conclude that Asf1N is functional for histone binding and
stimulation of histone deposition by CAF-I *in vitro*.

**Structure of Asf1N**

To explore the molecular basis of the many functional interactions of
Asf1N, we overexpressed the recombinant protein in *E.coli* and purified
it to homogeneity for structural studies (See Materials and Methods).
Contrary to full length Asf1, the purified truncated protein did not
aggregate in solution (data not shown) and crystals of Asf1N were
readily obtained by the hanging-drop vapor-diffusion method from a
variety of conditions using PEG3350 as the precipitant. The crystals
belong to space group C2 and contain one Asf1N monomer per asymmetric
unit.

We determined the structure of Asf1N to a resolution of 1.5Å using
Multiwavelength Anomalous Dispersion methods on crystals grown in high
concentrations of bromide ions (Figure 2A) (Table 1). The first 155
residues of Asf1 fold into an elongated (20Åx25Åx55Å) β-sandwich domain
topped by α-helices. The fold adopted by Asf1N belongs to the "switched"
immunoglobulin class of proteins (Figure 2B) \[33\], a broad group of
structures that in addition to immunoglobulins includes type III
fibronectins and cadherins \[34\]. A search of the structural database
\[33\] shows that Asf1N most closely superposes human type III
fibronectin (2fnb -- RMSD 2.7Å) \[35\] and the Rho guanine nucleotide
dissociation inhibitor RhoGDI (1rho- RMSD 2.9Å) \[36\].

An analysis of surface electrostatic potential, curvature, and amino
acid conservation highlights three regions of Asf1N that may be
important for its various functions (Figure 2C) (Figure 3). The first
and most striking of these is a concave groove on the "front" of the
molecule that is principally hydrophobic in character (Figure 3A). The
concavity of the groove is established by the curvature of the larger of
the two β-sheets, by the hairpin turn between strands β7 and β8, and by
the β4/β5 connector loop (Figure 2B). The groove is decorated with a
number of conserved, solvent exposed hydrophobic residues, including
V45, V92, L96 and Y112, and represents a potential protein-protein
interaction surface.

Flanking the groove lies a second region of interest, which is defined
by a strongly electronegative surface potential arising from a cluster
of acidic residues on β4 and β5 (D37, E39, D58 and D77). Many of these
show a high-degree of conservation (Figure 3B). Given the proximity of
this region to the hydrophobic groove and because electronegative
surfaces have previously been implicated as histone-binding determinants
\[37\], the acidic region of Asf1 represents a likely histone
interaction site. However, some residues in this region also appear to
be essential for stability of Asf1, because a double E39A, K41A mutation
resulted in nearly undetectable levels of Asf1 by immunoblotting of cell
extracts. Additionally, the Asf1E39A, K41A protein was non-functional
for *in vivo* HU sensitivity and silencing assays (data not shown).

The third region of interest lies on the other side of the hydrophobic
groove. This area, which lies near the "base" of Asf1N between strands
β1 and β10, contains a deep pocket near the N and C termini that is
lined with hydrophobic residues (Figure 3B). The pocket is connected to
a narrow channel that runs along the entire edge of the Asf1N molecule
(Figure 3). While narrower than the groove, the cleft appears
sufficiently wide to accommodate an additional β-strand from an
interacting protein to pair with β1. Indeed, it is worth noting that in
other "switched" Ig-fold proteins, a fourth strand is found at this
position \[34\]. The "top" of the molecule is decorated with two small
α-helical elements (α1 and α2) and a short 3-10 helix (α3) that appear
to be present in all organisms and to be moderately well conserved
(Figure 2C), providing another potential interaction region.

**The Hir1 binding site on Asf1**

In budding yeast, Asf1 and the Hir proteins physically interact and
function together to support the formation of silent chromatin and
regulate histone gene transcription \[15, 16\]. While budding yeast has
a single Asf1 protein, mammals retain two closely related proteins
termed Asf1a and Asf1b, along with a Hir1/Hir2 homolog known as HIRA. We
hypothesized that the Asf1-Hir protein interaction might be conserved in
evolution, and that we could use our structural data to define regions
of Asf1 important for this interaction.

To test these ideas, we first investigated the human Asf1a and HIRA
proteins, because we had observed interaction between these proteins in
two-hybrid experiments (data not shown); the interaction of Asf1b and
HIRA is under investigation. We confirmed a direct, stable interaction
between Asf1a and HA-epitope-tagged HIRA in coprecipitation experiments
using HA-epitope-tagged HIRA (Figure 4A). Alanine substituted mutant
human Asf1a proteins were then used to define potential binding sites
for HIRA on Asf1a. Mutation of either of two sets of contiguous amino
acids in human Asf1a, amino acids 36-37(ED to AA) and 62-64(VGP to AAA),
completely abolished Asf1a-HIRA interactions. In contrast, alanine
substitutions in at least four different regions of Asf1a have no effect
on HIRA binding, including: *a*) Asp13, which lies in the region between
β-strands 1 and 2, *b*) Glu49 and Glu51, which reside in the β4/β5
connector turn, *c*) Asp77, which is located at the end of strand β6,
and *d*) Glu121 and Glu124 of helix α2.

To determine whether the human Asf1a mutations might define a single
HIRA binding site, we mapped their position on the yeast Asf1N
structure. Indeed, both sets of mutations lie proximally to each other
on the surface of Asf1 (Figure 3B). In contrast, several other mutations
that did not affect HIRA binding were distributed elsewhere on the
tertiary structure. H36 and D37 are located on a loop immediately
preceding β4 while conserved residues V62, G63 and P64 define the end of
strand β5. These amino acids all lie adjacent to the hydrophobic groove
and D37 is a component of the acidic region (Figure 3B). A comparison of
Asf1 proteins shows that residues 62-64 (VGP) are nearly completely
identical throughout all eukaryotes (Figure 2C). P64 is one of two
cis-prolines present in Asf1N, the other being P15 at the turn between
the β1 and β2 strands. Along with G63, P64 is responsible for a sharp
turn in the loop immediately following β5. This arrangement anchors the
loop connecting β5 and β6 into a rigid, structured element. The loss of
this element would compromise the structural integrity of this region.
The VPG to AAA change is therefore likely to change the local backbone
fold as well as the surface characteristics. On the neighboring loop,
immediately adjacent to the VPG sequence, lie H36/D37. Of these two
residues, only D37 is highly conserved, suggesting that D37 is more
likely to play a key role in mediating Hir1 binding than H36. Further
characterization should determine whether Hir1 binding determinants
extend into either the acidic region or the conserved groove of Asf1.

To confirm these findings and examine their importance *in vivo*, we
recapitulated mutation of residues 36 and 37 in yeast Asf1. The
Asf1H36A,D37A mutant restored the DNA damage resistance to cells lacking
the chromosomal *ASF1* gene (Figure 4C), indicating that Asf1's role in
DNA damage repair and normal cellular growth was not affected by this
mutation. In contrast, in cells lacking both the *CAC1* and *ASF1*
genes, the mutated Asf1H36A,D37A protein was unable to restore telomeric
gene silencing (Figure 4D), even though normal levels of protein were
present (Figure 4B). Because the *HIR1* gene cooperates with *ASF1* to
contribute to silencing but does not have a role in DNA repair, these
data are consistent with the idea that the contribution of the Asf1 and
Hir1 proteins to silencing was specifically abolished by these
mutations.

**Comparison between nucleoplasmin and Asf1**

Although multiple histone binding and deposition proteins have been
identified to date \[3, 38, 39\], structural information is only
available for a few of these factors. Sequence analysis and secondary
structure prediction have revealed that several deposition proteins,
including two subunits of CAF-1 as well as HIRA-family proteins, contain
WD repeats characteristic of β-propeller structures \[40, 41\].
High-resolution crystallographic studies have provided a detailed view
of two Nucleoplasmin (Np)-type proteins \[37, 42, 43\], a second class
of histone binding and deposition factors that interact with H2A/H2B
dimers and are abundant in *Xenopus* and *Drosophila* embryos \[44,
45\].

Nucleoplasmin (Np), like Asf1, adopts a β-sandwich architecture.
However, the Np fold is based on a jelly-roll topology that is distinct
from the Ig-structure of Asf1N. Moreover, the functional unit of Np is a
dimer of pentamers, whereas Asf1N does not oligomerize in solution. The
different topologies and oligomerization states of Asf1N and Np-type
proteins may reflect to the different biological roles of these
proteins. For example, the multimeric arrangement of the Np protein core
may promote the generation of high-avidity, stable Np-histone complexes
to serve as embryonic histone storage elements, an important function of
Np proteins \[37\]. One similarity does exist between Np and the fungal
Asf1 homologs, namely the presence of extended, highly acidic C-terminal
tails. While not essential for function in either protein, these regions
nevertheless may act analogously to modulate electrostatic interactions
between the deposition proteins, histones, and the target DNA.
Interestingly, another yeast histone binding protein, Nap1, also
possesses a non-essential acidic tail, and recent evidence indicates
that these tails mediate the histone-binding specificity of Nap1 \[46\].

While additional mutagenesis and cocrystallization experiments will be
required to identify more fully the molecular determinants of histone
recognition for Asf1 and other histone deposition factors, the structure
of the Asf1 core region provides a starting point to define its role in
histone deposition and nucleosome assembly.

**Methods**

**Yeast strains and plasmids**

All yeast strains were in the W303 genetic background. The *asf1*Δ,
*cac1*Δ and *URA3-VIIL* alleles in this background have been described
previously \[16, 30\]. Telomeric silencing assays using the *URA3-VIIL*
reporter were also performed as previously described \[16, 29\]. For the
damage sensitivity and silencing assays, cells were grown 2-3 days at
30˚C before photographing, except for the FOA-Trp plates, which were
grown for 6 days. For *in vivo* complementation experiments, the *ASF1*
gene promoter was inserted into the *ARS/CEN, TRP1* vector pRS414
\[47\], and the full length *ASF1* gene or the *ASF1N* fragment was
inserted into this vector to allow for expression under control of the
natural promoter.

**Protein Expression and Purification**

Hexa-histidine tagged Rad53 was overexpressed by insertion of the
*RAD53* gene into pET28a (Novagen) to generate plasmid pHS4426. *E.
coli* BL21(pLysS) cells containing pHS4426 were grown to exponential
phase, and Rad53 expression was induced for 4 hours at 30˚C by the
addition of 1mM IPTG. Frozen cell pellets from a 2 L culture were
resuspended in 25mL Talon Binding Buffer (20mM Tris-HCl (pH8.0), 0.5 M
NaCl, 0.01% NP40, 10mM imidazole, 0.1mM PMSF, 0.5 μg/mL leupeptin, 0.7
μg/mL pepstatin, 1 μg/mL aprotinin, 1 μg/mL E64) and lysed by
sonication. The extract was clarified by centrifugation and incubated
with 1mL of Talon immobilized metal resin (Clonetech) for 1hr at 4˚C.
The resin was washed in batch twice with 10 bed volumes of Talon Binding
Buffer, and Rad53 was then eluted by addition of 10mL Talon Elution
Buffer (Buffer 20mM Tris-HCl (pH8.0), 0.5M NaCl, 0.01% NP40, 100mM
imidazole, 0.1mM PMSF). Rad53 was dephosphorylated by the addition of 4
Units of Lambda Phosphatase (New England Biolabs) per µg of Rad53 in the
buffer recommended by the manufacturer containing 0.2mM MnCl2. After
incubating 40min at 30˚C the protein was dialyzed into Talon Binding
Buffer and purified away from the phosphatase by chromatography on Talon
resin as above. Rad53 was stored in 25mM Tris-HCl, pH 7.5, 0.5 M NaCl,
1mM EDTA, 0.01% NP40 10% glycerol, 1mM DTT, 0.1mM PMSF.

C-terminally FLAG-epitope tagged Asf1N (Asf1N-FLAG) was expressed by
insertion of *ASF1* codons 1-155 followed by two FLAG epitopes into
pET28a (Novagen). Asf1N-FLAG was purified by metal affinity
chromatography followed by S-200 gel filtration chromatography
(Pharmacia) and Poros HQ (Roche) ion exchange chromatography.
C-terminally FLAG-epitope tagged, full length Asf1 was expressed as
described \[16\] and purified as described above for the Asf1N-FLAG
protein.

For crystallization, Asf1N was expressed in *E. coli* using a modified
pET expression vector encoding an N-terminal hexa-histidine tag followed
by a TEV protease cleavage site (N. Pokala and T. Handel, personal
communication). The TEV cleavage site was engineered such that the
N-terminus after cleavage is H2N-G-A-S-, with the S residue representing
the second amino acid of natural Asf1. Asf1N was purified from cell
lysates by metal affinity chromatography on Talon resin according to
manufacturer's instructions (Clonetech). The histidine tag was cleaved
from Asf1N by hexa-histidine tagged TEV protease digestion at 25oC for
16 h \[48\], and the TEV was subsequently removed by metal affinity
chromatography. Cleaved Asf1N was further purified by gel filtration
chromatography on a Pharmacia S-200 column; peak fractions displayed an
apparent molecular weight of 20 kDa, consistent with an Asf1N monomer.
Purified Asf1N was concentrated to 5-10 mg/mL using a Centriprep
concentrator (Millipore).

**Protein Binding and Histone Deposition Assays**

Histone (H3.H4)2 tetramers were purified from HeLa cell nuclei as
described \[49\]. 75 pmol of histones (H3.H4)2 were preincubated with an
equimolar amount of either Flag-Asf1 or Flag-Asf1N for 30 minutes at
30˚C in 30 μL of histone binding buffer (30mM Hepes-NaOH (pH 7.5), 5%
glycerol, 100mM NaCl, 0.1% Triton X100, 0.1 mM EDTA, 0.1 mg/mL BSA). The
reactions were then incubated for 1 hour at room temperature in binding
buffer with anti-Flag M2 antibody agarose beads (Sigma) in a final
volume of 200 μL. Beads were washed four times with 1 mL of wash buffer
(30 mM Hepes-NaOH (pH 7.5), 5% glycerol, 0.1% Triton X-100, 0.1 mM EDTA,
0.1 mg/mL BSA, and either 250 mM or 500 mM NaCl). Proteins were eluted
with sample buffer and analyzed on a 17% SDS-PAGE gel stained with
Coomassie Blue R250.

The binding and washing buffer for the Asf1-Rad53 interaction
experiments was the same used for histone binding described above,
except that the NaCl concentration was 100mM. Binding was performed in a
total volume of 40µL for 30 minutes at 30˚C prior to immunoprecipitation
and washing as described above, using the 100mM NaCl-containing buffer.

Histone deposition during *in vitro* DNA replication by CAF-I was
assayed as described \[13, 16, 30\]. Complexes of purified Asf1 or Asf1N
with histones H3 and H4 were formed by mixing at a four-to-one molar
ratio of Asf1 to histone tetramer in binding buffer (30 mM Hepes-NaOH
(pH 7.5), 100 mM NaCl, 0.1% Triton X100, 0.1 mM EDTA, 0.1 mg/ml BSA) and
incubation at 30oC for 30 minutes. Replication reactions were initiated
and incubated at 30oC for twenty minutes prior to addition of
Asf1/histone complexes, and reactions were continued for seventy
minutes.

Co-immunoprecipitation assays with *in vitro*-translated human Asf1a and
HIRA were performed as described \[50\]. Protein complexes were washed
five times in NETN (20mM Tris-HCl pH8.0, 100mM NaCl, 1mM EDTA, 0.5%
NP40).

**Crystallography, Data Collection and Refinement**

Prior to crystallization, concentrated Asf1N was dialyzed into 10 mM
Hepes-NaOH (pH 7.5), 50 mM NaCl and 1 mM TCEP at 4˚C. Initial
needle-like crystals were grown at 18˚C in sitting drops by mixing 0.8
µl of a reservoir solution containing 100 mM NH4F, 100mM NaBr and 20%
PEG 3350 with 0.8 µl dialyzed Asf1N. Small crystals were harvested,
diluted 100x in well solution and used for macroseeding. Large single
crystals plate-like crystals (150x100x20) were grown in hanging drops by
mixing 0.9 µl well solution (0.85-1.0 M NaBr and 17% PEG 3350), 0.9 µl
Asf1N and 0.2 µl of seed stock. Single crystals were harvested into
cryo-protectant solution (10 mM HEPES-NaOH (pH 7.5), 20% PEG 3350, 25%
PEG400, 1M NaBr, 50 mM NaCl and 1mM TCEP) and flash-frozen in liquid
nitrogen.

MAD data were collected using the Advanced Light Source Beamline 8.3.1
at Lawrence Berkeley National Laboratory (Table 1). Diffraction data
were processed with HKL2000 \[51\]. The crystals contain one molecule in
the asymmetric unit (25% solvent) and belong to the space group C2
(a=94.36Å, b=31.7Å, c=53.6Å, α=γ=90°, β=124.6°. A total of nine Br -
sites were located using SOLVE \[52\] and an initial model was built
into the experimental electron density maps using RESOLVE \[52\].
Subsequent model building was carried out using O \[53\]. Refinement was
performed using REFMAC5 and ARP \[54\]. The final model accounts for all
residues in the Asf1N sequence, 92.7% of which fall in the most favored
regions of Ramachandran space as determined by PROCHECK \[55\].

**Coordinates**

Coordinates for Asf1N have been deposited in the Protein Data Bank and
are awaiting an accession number.

**Acknowledgements**

This work was supported by Department of Energy funds awarded to P.D.K.
and administered through the Lawrence Berkeley National Laboratory,
National Science Foundation Grant MCB-9982909 (P.D.K.), National
Institutes of Health grant GM55712 (P.D.K.), and NIH grant GM055583 (S.
J. B). J.M.B gratefully acknowledges support from the G. Harold and
Leila Y. Mathers Charitable Foundation. The work of PDA is supported by
NIH R01 GM62281, DAMD17-02-1-0726 and a Leukemia and Lymphoma Society
Scholar award.

**Figure Legends**

**Figure 1. Yeast Asf1N protein is functional *in vivo* and *in vivo*.**

\(A\) Asf1N confers DNA damage resistance *in vivo*. Yeast strains of
the indicated genotypes were transformed with low copy plasmids
expressing full length Asf1, Asf1N, or an empty vector as indicated.
Strains were grown to logarithmic phase, serially diluted, and spotted
onto synthetic complete media lacking tryptophan (-Trp) to score total
plasmid-containing cells, or onto media containing 0.004% methyl
methanesulfonate (MMS) or 0.1M hydroxyurea (HU).

\(B\) Asf1N promotes chromatin-mediated gene silencing at telomeres.
Cells of the indicated genotypes containing an *URA3-VIIL* telomeric
reporter gene were transformed with plasmids expressing full length
Asf1, Asf1N, or an empty vector as indicated. Strains were grown to
logarithmic phase, serially diluted, and plated as indicated onto --Trp
media to assess total cell number or onto FOA-Trp to measure silencing.

\(C\) Asf1N binds to histones H3/H4. Recombinant, FLAG epitope-tagged
Asf1 or Asf1N were incubated with histones H3/H4, immunoprecipitated
with anti-FLAG antibodies coupled to a solid resin, and the precipitates
were washed as indicated. Retained proteins were visualized by Coomassie
staining of a 17% SDS-PAGE gel. (D) Asf1N binds to Rad53. 15 pmol (lanes
1-3) or 30 pmol (lanes 4-6) of recombinant, dephosphorylated Rad53 was
incubated with no protein (lanes 1 and 4) or 15 pmol FLAG epitope-tagged
Asf1 (lanes 2 and 5) or Asf1N (lanes 3 and 6) and immunoprecipitated
with anti-FLAG antibodies. Input lanes contain 15 pmol of each protein
(lanes 7-9). Proteins were visualized by Coomassie staining of a 15%
SDS-PAGE gel.

\(E\) Asf1N stimulates the replication-coupled histone deposition
activity of CAF-I. Where indicated, a substoichiometric amount of
recombinant yeast CAF-I (12 ng, \[13\]) was added. Preformed
Asf1.(H3.H4)2 or Asf1N.(H3.H4)2 complexes were titrated into the
reactions at 0.44, 0.88, or 1.75 pmol of histone tetramer in lanes 3-5,
6-8, 9-11 and 12-14, respectively. Stimulation of histone deposition is
indicated by the increased proportion of supercoiled DNA, as observed in
lanes 5 and 11.

**Figure 2.** **Backbone structure and amino acid conservation of
Asf1N.**

\(A\) A portion of the 1.5Å resolution experimental electron density map
contoured at 1σ from the region around strand β3.

\(B\) Stereo view of a ribbon diagram of Asf1N illustrating the
secondary structure elements. This view faces the hydrophobic cleft
between strands β1 and β10 (see Figure 3).

\(C\) Comparison of Asf1 protein sequences and structure. Secondary
structure elements are labeled over sequences and match those indicated
in panel B. The degree of evolutionary conservation as determined using
clustalX \[56\], is shown as a gradient of red, orange, yellow, and
white boxes, with red being most highly conserved and white being
non-conserved. The purple triangle at the end of the sequence marks the
natural termination point of the *Leishmania* Asf1 protein. Panels A and
B were rendered using PYMOL \[57\]. Panel C was generated using ALSCRIPT
\[58\]. Human-1 refers to the Asf1a protein (accession number CAB43363)
and Human-2 refers to the Asf1b protein (accession number NP_060624).

**Figure 3. Surface properties of Asf1N.**

\(A\) Conservation of residues on the front face. Left panel: Stereo
view of front face of Asf1N. The groove formed by the concave front
sheet of beta strands is labeled, as is the hydrophobic cleft between
strands β1 and β10 that runs along the side of the protein. Coloration
scheme is as in Figure 2C. Right panel: Ribbon diagram of Asf1N
secondary structure viewed from the same angle as the left panel.

\(B\) Electrostatic and curvature properties of Asf1N. The middle
diagram shows the surface charge of the front view of the protein,
viewed from the same angle as Figure 2A. Negative charge is shown in
red, positive charge in blue. On the left, the diagram was turned 90° to
the right to reveal the negatively charged surface formed largely by
strands β4 and β5. On the right, the middle diagram was turned 90° to
the left, and the concavity of the surface is highlighted with convex
surfaces in green and concave surfaces in black. The deep hydrophobic
cleft between strands β1 and β10 is labeled. Surface panels were
generated with GRASP \[59\] and ribbon diagrams with PYMOL \[57\]. Amino
acid conservation was mapped onto the protein surface in panel A using
CONSURF \[60\]. Adjacent sets of residues implicated in Hir protein
interaction are highlighted: residues 36-37 in cyan and 62-64 in violet.

**Figure 4. Surface residues 36 and 37 are required for the human
Asf1a-HIRA protein interaction and for Asf1-mediated telomeric silencing
but not DNA damage resistance in yeast.**

\(A\) Human HA-tagged HIRA (lanes 2-8), wild-type (WT) human ASF1a
(lanes 1-2) and a series of mutant human ASF1a proteins (lanes 3-8) were
synthesized by *in vitro* translation in the presence of 35S-methionine.
Proteins were immunoprecipitated with an antibody to HA (12CA5) and
bound proteins were fractionated by SDS-PAGE and visualized by
fluorography. The bottom panel shows the 1/5th of the total input ASF1a.
ASF1a mutants: ED36-37AA (lane 3), VGP62-64AAA (lane 4), D13A (lane 5),
ESE49-51ASA (lane 6), D77A (lane 7), ELRE121-124ALRA (lane 8).

\(B\) Immunoblot of total cell extracts from strains analyzed in Figure
4D. Two independent isolates of the *asf1-H36D37-AA* cells were tested,
in which the *H36D37-AA* mutation was made in the context of full-length
Asf1. 5 micrograms total cell protein was probed with antibodies raised
against Asf1 (top panel). The same blot was reprobed with antibodies
that recognize PCNA as a loading control(bottom panel).

\(C\) Wild-type HU sensitivity of *asf1-H36D37-AA* cells. Two
independent isolates of the *asf1-H36D37-AA* cells were grown and plated
as described in Figure 1A.

\(D\) Telomeric silencing defect of *cac1 asf1-H36D37-AA* cells. Cells
were grown and plated as described in Figure 1B.

**Table 1. Structure Determination Statistics.**

**Data Collection**

  --------------------------------- ---------------------- --------------------
                                    [Remote]{.underline}   [Peak]{.underline}

  --------------------------------- ---------------------- --------------------

  --------------------------------- ------------------ ------------------
  Radiation Å                       0.956              0.920

  --------------------------------- ------------------ ------------------

  --------------------------------- ------------------ ------------------
  Resolution Å                      50-1.5             50-1.6

  --------------------------------- ------------------ ------------------

  --------------------------------- ------------------ ------------------
  Completeness % (last shell)       94.0 (82.4)        91.6 (61.9)

  --------------------------------- ------------------ ------------------

  --------------------------------- ------------------ ------------------
  Overall I / σ (last shell)        13.45 (2.6)        11.3 (1.7)

  --------------------------------- ------------------ ------------------

  --------------------------------- ------------------ ------------------
  Rsym\* % (last shell)             5.3 (23.9)         6.7 (28.8)

  --------------------------------- ------------------ ------------------

**Phasing**

  ----------------------- ------------ -----------------------------------
  Atom                                 Br

  ----------------------- ------------ -----------------------------------

  ----------------------- ------------ -----------------------------------
  No. sites                            9

  ----------------------- ------------ -----------------------------------

  ----------------------- ------------ -----------------------------------
  Mean Figure of Merit                 0.39

  ----------------------- ------------ -----------------------------------

**Structure Refinement**

  ---------------------- -------- -------------------------- ------------
  Resolution range (Å)   30-1.5   Overall reflections (last  19972 (1343)
                                  shell)                     

  ---------------------- -------- -------------------------- ------------

  ---------------------- -------- -------------------------- ------------
  No. nonhydrogen atoms  1414     Test reflections (last     1082 (85)
                                  shell)                     

  ---------------------- -------- -------------------------- ------------

  ---------------------- -------- -------------------------- -------------
  No. protein atoms      1235     Rwork/Rfree\*\*(%)         19.96/24.14

  ---------------------- -------- -------------------------- -------------

  ---------------------- -------- -------------------------- -------------
  No. ligand atoms       9        Rmsdbonds/angles           0.009/1.078

  ---------------------- -------- -------------------------- -------------

+----------------------+-------+--------------------------+-----------+
| No. water atoms      | 168   | Ramachandran % (no.      | 92.7      |
|                      |       | res.)                    | (127)     |
|                      |       |                          |           |
|                      |       | most favored             | 6.6 (9)   |
|                      |       |                          |           |
|                      |       | allowed                  | 0.7 (1)   |
|                      |       |                          |           |
|                      |       | gen. allowed             |           |
+----------------------+-------+--------------------------+-----------+

\*Rsym = ΣΣj\|Ij - \<I\>\|/ΣIj, where Ij is the intensity measurement
for reflection j and \<I\> is the mean intensity for multiply recorded
reflections.

\*\*Rwork, free = Σ\|\|Fobs\| - \|Fcalc\|\|/Σ\|Fobs\|, where the working
and free R factors are calculated using the working and free reflection
sets, respectively. The test reflections (5.2% of the total) were held
aside throughout refinement.

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