Structure of C-terminal Tandem BRCT Repeats of Rtt107
Protein Reveals Critical Role in Interaction with
Phosphorylated Histone H2A during DNA Damage Repair*□
Xinxin Li, Kaixian Liu, Fudong Li, Juncheng Wang, Hongda Huang, Jihui Wu1, and Yunyu Shi2
TechnologyofChina,Hefei,Anhui 230027, China
Background: Rtt107 can be recruited to chromatin during the DNA damage response.
Results: Structures of C-terminal Rtt107 alone and in a complex with ?H2A were determined.
Conclusion: Mutagenesis studies indicated that the phosphorylation-dependent interaction between Rtt107 and ?H2A is
important for the function of Rtt107.
Rtt107 (regulator of Ty1 transposition 107; Esc4) is a DNA
repair protein from Saccharomyces cerevisiae that can restore
stalled replication forks following DNA damage. There are six
BRCT (BRCA1 C-terminal) domains in Rtt107 that act as bind-
Rtt107 binding partners have been identified, including Slx4,
mosome) protein complex. Rtt107 can reportedly be recruited
to chromatin in the presence of Rtt101 and Rtt109 upon DNA
damage, but the chromatin-binding site of Rtt107 has not been
identified. Here, we report our investigation of the interaction
nal tandem BRCT repeats (BRCT5-BRCT6) of Rtt107. The crys-
tal structures of BRCT5-BRCT6alone and in a complex with
?H2A reveal the molecular basis of the Rtt107-?H2A interac-
tion. We used in vitro mutagenesis and a fluorescence polariza-
tion assay to confirm the location of the Rtt107 motif that is
crucial for this interaction. In addition, these assays indicated
vivo phenotypic analysis in yeast demonstrated the critical role
of BRCT5-BRCT6and its interaction with ?H2A during the
ular mechanism by which Rtt107 is recruited to chromatin in
response to stalled DNA replication forks.
and duplicate billions of DNA base pairs. Three-dimensional
DNA structures, such as replication forks that are formed dur-
ing DNA synthesis, are very sensitive to both endogenous and
exogenous insults (1). The repair of these lesions generally
occurs in a stepwise manner. In Saccharomyces cerevisiae, the
checkpoint kinase Mec1 is recruited to the break sites early
during the damage response (2). Mec1 then phosphorylates a
variety of proteins in the DNA replication and repair machin-
Rtt107 is one substrate of Mec1 phosphorylation and was
poson mobility (5). The Rtt107? mutant is very sensitive to a
wide spectrum of replication stress-inducing agents, such as
the DNA-alkylating agent methyl methanesulfonate (MMS),3
the nucleotide reductase inhibitor hydroxyurea (HU), and the
topoisomerase I poison camptothecin (CPT) (6–8). Further-
absence of DNA-damaging agents (9). Rtt107 is required for
normal DNA synthesis and to restart stalled replication forks
during recovery from DNA damage in S phase (6, 8). In addi-
repair (10, 11).
Structurally, Rtt107 contains six BRCT homology domains.
There are four tandem BRCT domains at the N terminus of
is a phosphoprotein interaction module frequently found in
proteins involved in the DNA damage response, cell cycle con-
trol, and checkpoint-mediated DNA repair (13–16). It is
believed that the multiple BRCT domains of Rtt107 create a
during DNA damage repair (17). Consistent with this role,
Rtt107 interacts with several repair proteins, such as the struc-
the Smc5/6 complex (10, 11, 19). The N-terminal BRCT
* This work was supported by National Basic Research Program of China 973
Program Grants 2011CB966302 and 2011CB911104 and by Chinese
National Natural Science Foundation Grants 30830031 and 31170693.
SThis article contains supplemental Figs. S1 and S2.
The atomic coordinates and structure factors (codes 3T7I, 3T7J, and 3T7K) have
been deposited in the Protein Data Bank, Research Collaboratory for Struc-
3The abbreviations used are: MMS, methyl methanesulfonate; HU,
hydroxyurea; CPT, camptothecin; r.m.s.d., root mean square deviation;
Rtt107-C, Rtt107 C terminus.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 12, pp. 9137–9146, March 16, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc.Published in the U.S.A.
by guest on September 23, 2015
domains of Rtt107 interact with Slx4 and the Smc5/6 complex
(7, 10), but the binding partners of the C-terminal BRCT
repeats during DNA damage repair have not yet been
In this study, we performed sequence alignment and found
that only the C-terminal tandem BRCT repeats (BRCT5-
BRCT6) of Rtt107 contain phospho-recognition modules (20,
21). These data suggest that BRCT5-BRCT6may interact with
unidentified phosphoproteins during DNA damage repair.
in the presence of Rtt101 and the acetyltransferase Rtt109 in
response to stalled replication forks. However, the chromatin-
binding site of Rtt107 is poorly understood. An important clue
is that Brc1, the fission yeast homolog of Rtt107, forms ?H2A-
dependent nuclear foci in the presence of DNA damage (20).
ing to ?H2A.
To explore the structure and function of the C-terminal tan-
dem BRCT repeats of Rtt107 during DNA damage repair, we
solved the crystal structures of BRCT5-BRCT6alone and in a
firmed the phosphorylation-dependent interaction between
tional studies reveal the structural basis of the Rtt107-?H2A
interaction and shed new light on the interaction network of
Rtt107 during DNA damage repair.
Cloning, Expression, and Purification of Protein—A DNA
fragment of Rtt107-C (residues 820–1070) was amplified from
yeast genomic DNA (S. cerevisiae S288C) by PCR. This frag-
ment was ligated into plasmid pGEX-4T-1 (GE Healthcare)
using NdeI/XhoI, yielding plasmid pGEX-Rtt107-C. The
Rtt107-C mutations for L909M, L1028M, T842A, and K887M
were introduced using the MutanBEST kit (Takara Co.). The
Protein expression was induced at A600? 0.8–1.2 with 0.2 mM
isopropyl ?-D-thiogalactopyranoside, and the cells were grown
at 16 °C for 18 h. The proteins were purified by GST-glutathi-
one affinity chromatography and eluted with on-resin throm-
further purified by Superdex 75 gel filtration chromatography
(GE Healthcare) in buffer A (500 mM NaCl and 20 mM Tris-
B (50 mM NaCl and 10 mM Tris-HCl, pH 7.5) and concentrated
to 20–40 mg/ml.
To prepare the SeMet-derivatized protein, Rtt107-C con-
taining the L909M and L1028M mutations was expressed in
E. coli strain B834 (Novagen) using LR (a minimal medium
supplemented with SeMet and six amino acids (leucine, isoleu-
cine, valine, phenylalanine, lysine, and threonine). The SeMet-
derivatized protein was purified by a procedure similar to the
one used to purify the native proteins.
Crystallization and Data Collection—Crystals of both native
Rtt107-C and the SeMet-derivatized L909M/L1028M mutant
were grown at 283 K by mixing 1 ?l of 20 mg/ml protein in
buffer B with 1 ?l of well solution 1 (17.5% (w/v) mPEG 2000,
200 mM NaCl, and 100 mM HEPES, pH 7.0) using the hanging
drop vapor diffusion method. Single crystals were obtained
after 2 days.
All peptides used in this study were synthesized at GL
Biochem (Shanghai) Ltd. The complex between Rtt107-C and
Rtt107-C protein with the ?H2A phosphopeptide (ATKAp-
SQEL) in buffer B at a 1:1.5 protein/phosphopeptide molar
For data collection, the crystals were flash-frozen in liquid
nitrogen after being transferred into a cryoprotectant solution
composed of 80% mother liquor and 20% glycerol. All single-
crystal x-ray diffraction data were collected at the Shanghai
Synchrotron Radiation Facility (SSRF) using beamline BL17U.
The multiple-wavelength anomalous dispersion data set
(?peak? 0.9805 Å, ?inflection? 0.9807 Å, and ?remote? 0.9506
tein at 100 K. X-ray data reduction and scaling were performed
with the HKL2000 suite (23).
X-ray Structure Determination and Refinement—All four
(two selenomethionines per C-terminal polypeptide) were
located and refined with SOLVE (24) and a three-wavelength
SeMet multiple-wavelength anomalous dispersion data set.
The initial phases were calculated by RESOLVE (25) with a
resolution ranging between 30 and 2.30 Å, and an initial model
was automatically built. The model was further built and
by manual model correction until the crystallography R-factor
The structure of the SeMet-labeled Rtt107-C L909M/L1027M
mutant was used as an initial search model for determining the
native structure of Rtt107-C by a standard molecular replace-
ment method in the PHENIX package (28). The final crystal-
lography R-factor and the free R-factor of the native Rtt107-C
structure are 20.3 and 25.4%, respectively. The structure of the
complex between Rtt107-C and ?H2A was determined using
the native Rtt107-C structure as the initial search model in the
PHENIX package (28). The complex structure was further
refined by procedures similar to those described above. Details
regarding the data collection and processing of these crystal
structures are presented in Table 1.
Fluorescence Polarization Assay—The FITC probe was con-
jugated to ?H2A and H2A using a chemical reaction described
in standard protocols. The labeled peptides were purified with
an FPLC column. Fluorescence polarization assays were per-
formed in buffer B at 293 K using a SpectraMax M5 microplate
reader system. The wavelengths of fluorescence excitation and
emission were 490 and 520 nm, respectively. Each well of a
96-well plate contained 100 nM FITC-labeled peptide and dif-
ferent amounts of Rtt107-C or the Rtt107-C mutant (concen-
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trations ranged between 0 and 200 ?M) in a final volume of 200
?l. For each assay, peptide-free controls (Rtt107-C or the
ization (P, in millipolarization (P/1000) units) was calculated
according to Equation 1.
P ?I?? I?
The fluorescence polarization change (?P, in millipolarization
(P/1000) units) was fit to Equation 2.
?P ??Pmax? ?Rtt107?
CD Spectroscopy—The CD spectra of Rtt107 and its mutant
were recorded at 298 K on a Jasco J-810 spectropolarimeter.
nm using a 0.1-cm path length cell and 0.2 mg/ml protein in
PBS, pH 7.4. A buffer-only sample was used as the reference.
The molar ellipticities ([?]) were plotted against the wave-
Yeast Strains, Plasmids, and Experiments—To construct
pRS316-Rtt107, the entire Rtt107 open reading frame plus 584
bp of upstream and 100 bp of downstream genomic sequence
were cloned at the BamHI/XhoI sites of pRS316. The pRS316-
Rtt107 mutations, including ?BRCT5-BRCT6and T842A plus
K887M, were introduced using the MutanBEST kit. The
pRS316-Rtt107 plasmids encoding wild-type or mutant con-
structs were transformed into yeast strain ?Rtt107 (BY4742,
Invitrogen) by electroporation (29). For functional studies of
Rtt107, the cells were grown to the exponential phase and then
2% peptone, and 2% dextrose) plates containing 0.03% MMS, 5
?g/ml CPT, 50 mM HU, or no DNA stress agent.
Crystal Structure of C-terminal BRCT Domains of Rtt107—
The crystal structures of Rtt107-C (residues 820–1070;
BRCT5-BRCT6) were determined by multiple-wavelength
anomalous dispersion phasing and molecular replacement
statistics are summarized in Table 1. There are two copies of
Rtt107-C packed against each other to compose one asymmet-
ric unit of the crystal. As shown in Fig. 1, the overall structure
illustrates the tandem BRCT repeats as expected. Surprisingly,
the N terminus of BRCT5contains an additional helix, named
?N. From the structure, we found that some residues (Ala-822,
Ile-825, Leu-826, Phe-829, and Leu-832) of the ?N helix and
N-terminal loop were packed together with residues of the ?1
helix (Val-852, Glu-855, and Ile-856) and the ?3 helix (Ile-913)
of BRCT5(Fig. 1B). These three ? helices form a three-helix
bundle stabilized predominantly by hydrophobic interactions.
We were not able to obtain any stable proteins without the ?N
helix, presumably due to its essential position within the three-
helix bundle. Despite the N-terminal extension, BRCT5con-
sists of a four-stranded parallel ? sheet (?1, ?2, ?3, and ?4)
(?2) on the other side; this structure is similar to other BRCT
BRCT6, similar to BRCT5, shows a remarkable variation to
the canonical BRCT domain. The ? sheet core consists of four
strand (?5?), which is unique among the BRCT domains. The
BRCT6sequence is not homologous to any other structures
a ? 59.24, b ? 74.64, c ? 129.08 Å;
? ? 90°, ? ? 90°, ? ? 90°
a ? 34.36, b ?
62.18, c ? 65.80 Å;
? ? 86.64°, ? ?
75.38°, ? ? 73.98°
a ? 34.16, b ?
59.23, c ? 85.89 Å;
? ? 78.36°, ? ?
89.68°, ? ? 73.17°
No. of reflections
No. of atoms
aValues in parentheses are for the highest resolution shell.
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recorded in the Protein Data Bank, further defining its unique
role among BRCT domains. There are two disordered regions
(residues 922–926 and 1007–1014) on the outer face of these
two BRCT domains that are missing in the electron density
The two BRCT domains adopt a head-to-tail arrangement,
core BRCT structural elements and the interdomain linker.
Helix ?2 packs against helices ?1? and ?3?, forming a helix
bundle with helix ?L1. The interface is stabilized by the hydro-
phobic interactions of residues within the helix bundle. Polar
contacts between Arg-884 and Asn-1044 in addition to the
hydrophobic interactions between Lys-942 and Leu-1053 fur-
ther stabilize the interface (Fig. 1C). The tandem BRCT
domains form a rigid body due to the stable interface between
Structural Comparison with Other Tandem BRCT Repeats—
There is very low sequence identity between BRCT5-BRCT6of
Rtt107 and the tandem BRCT repeats of other proteins. (There
is only 19% identity with the homolog Brc1 (supplemental Fig.
S1).) A structural comparison may further our understanding
of the BRCT domains of Rtt107. We used the BRCT5-BRCT6
structure of Rtt107 as the search model in the Dali server (30).
Not surprisingly, the root mean square deviation (r.m.s.d.)
between BRCT5-BRCT6of Rtt107 and other tandem BRCT
shows the highest similarity to the four tandem BRCT repeats
of Brc1 (fission yeast homolog of Rtt107, r.m.s.d. of 3 Å) (20),
MDC1 (r.m.s.d. of 3.4 Å) (31), BRCA1 (r.m.s.d. of 3.4 Å) (32)
and 53BP1 (r.m.s.d. of 3.8 Å) (33). All of these proteins play
important roles in DNA damage repair. As shown in Fig. 2, the
positions and orientations of the secondary structures in the
C-terminal tandem BRCT repeats of Rtt107 deviate consider-
ably from those of the homolog Brc1. In Rtt107, the ?1 helix
tilts ?30° compared with the ?1 helix in Brc1, and the ?3 helix
extends 6 Å longer along its helical axis (Fig. 2B) than the same
structure in Brc1. These changes cause these two helices to
pack more tightly with the ?N helix in Rtt107. The orientation
of the main ?2? helix in the C terminus of the BRCT domain
changes as much as 80° from the main helix in Brc1 (Fig. 2C).
Another remarkable difference is that the linker regions of
BRCT5-BRCT6in Rtt107 are shorter and packed more tightly
than the other three BRCT repeats (supplemental Fig. S2). A
previous study has shown that the linker regions between
BRCT repeats are highly variable both in sequence and in
length (34). Therefore, BRCT linker regions may provide spec-
ificity for binding interactions. For example, the ? hairpin of
53BP1 is involved in the interaction with the tumor suppressor
protein p53 (35, 36). It would be interesting to investigate the
function of the short linker region in BRCT5-BRCT6of Rtt107.
Williams et al. (20) found that there are conserved electro-
FIGURE 1. Crystal structure of BRCT5-BRCT6of Rtt107. A, overall three-dimensional structure of BRCT5-BRCT6. The two BRCT domains are colored cyan and
green. The linker region is orange. Disordered regions are shown as dotted lines. B, the three N-terminal helices (?N, ?1, and ?3) of BRCT5form a helix bundle.
The labeled residues are involved in hydrophobic interactions within the helix bundle. C, interface between BRCT5and BRCT6. The interactions between the
labeled residues render the tandem BRCT repeats a rigid body.
9140 JOURNALOFBIOLOGICALCHEMISTRY VOLUME287•NUMBER12•MARCH16,2012
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region in Rtt107 has been changed (Fig. 3). It contains several
hydrophobic residues, and these residues split the electronega-
tive patch of Rtt107. Whether Rtt107 can interact with the sur-
face of histone core particles remains a question.
BRCT5-BRCT6of Rtt107 Binds to ?H2A—Rtt107 is believed
to act as a scaffold during DNA damage repair and can be
recruited to chromatin in the presence of Rtt109 and Rtt101 in
tin-binding site of Rtt107 has not been identified. Tandem
ysis indicates that there are phospho-recognition modules in
the fifth BRCT domain of Rtt107; the C1 (TG) motif is located
this region forms a positively charged binding pocket (Fig. 4A).
C-terminal phosphorylation of H2A (?H2A) is very important
during DNA damage repair and chromatin packing (38), and
the docking of Brc1 to ?H2A is critical for the chromatin-spe-
cific response to replication-associated DNA damage (20).
Thus, Leung et al. (10) proposed ?H2A as a possible target of
Rtt107. To test whether BRCT5-BRCT6in Rtt107 can bind
gated FITC to the N terminus of this peptide. A fluorescence
polarization binding assay showed that BRCT5-BRCT6in
is ?8 ?M, which is similar to other tandem BRCT repeats. The
mutations within the C1 and C2 motifs (T842A and K887M)
block most of the interaction, and the CD spectra indicate that
these mutations do not alter the overall protein structure (Fig.
4C). No interaction was detected between Rtt107 and unphos-
H2A in a phosphorylation-dependent manner.
Complex Structure of BRCT5-BRCT6of Rtt107 with ?H2A—
To investigate the molecular basis of the interaction between
Rtt107 and ?H2A, we co-crystallized BRCT5-BRCT6of Rtt107
with the ?H2A tail. The structure (Protein Data Bank 3T7K)
FIGURE 2. Structural comparison between Rtt107 and Brc1. A, structural
comparison of BRCT5-BRCT6of Rtt107 (green) with BRCT5-BRCT6of Brc1 (tan;
Protein Data Bank code 3L40). Structural alignment was carried out with the
helix in Brc1 (tan).
FIGURE 3. Comparison of surface electrostatic potentials between Rtt107 and Brc1. A, electrostatic surface of Rtt107 (black ellipse) corresponding to the
predicted nucleosome-binding site of Brc1. B, the black ellipse indicates the predicted nucleosome-binding site of Brc1 (20). C, sequence alignment of Rtt107
and Brc1. The red arrowheads highlight the acidic residues that belong to the predicted surface in Brc1.
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