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-
by guest on September 23, 2015
J. Biol. Chem.
Wang, Hongda Huang, Jihui Wu and Yunyu
Xinxin Li, Kaixian Liu, Fudong Li, Juncheng
Histone H2A during DNA Damage Repair
Role in Interaction with Phosphorylated
Repeats of Rtt107 Protein Reveals Critical
Structure of C-terminal Tandem BRCT
Protein Structure and Folding:
doi: 10.1074/jbc.M111.311860 originally published online January 19, 2012
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