RPA Accumulation during Class Switch
Recombination Represents 50–30DNA-End Resection
during the S–G2/M Phase of the Cell Cycle
Arito Yamane,1Davide F. Robbiani,3Wolfgang Resch,1Anne Bothmer,3,7Hirotaka Nakahashi,1Thiago Oliveira,3
Philipp C. Rommel,3Eric J. Brown,5Andre Nussenzweig,6Michel C. Nussenzweig,3,4,* and Rafael Casellas1,2,*
1Genomics & Immunity, NIAMS
2Center of Cancer Research, NCI
National Institutes of Health, Bethesda, MD 20892, USA
3Laboratory of Molecular Immunology
4Howard Hughes Medical Institute
The Rockefeller University, New York, NY 10065, USA
5Abramson Family Cancer Research Institute and Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania,
Philadelphia, PA 19104, USA
6Laboratory of Genome Integrity, NCI, National Institutes of Health, Bethesda, MD 20892, USA
7Present address: Cancer Genetics Program, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA
*Correspondence: firstname.lastname@example.org (M.C.N.), email@example.com (R.C.)
Activation-induced cytidine deaminase (AID) pro-
motes chromosomal translocations by inducing
DNA double-strand breaks (DSBs) at immunoglob-
ulin (Ig) genes and oncogenes in the G1 phase. RPA
is a single-stranded DNA (ssDNA)-binding protein
that associates with resected DSBs in the S phase
and facilitates the assembly of factors involved in
homologous repair (HR), such as Rad51. Notably,
RPA deposition also marks sites of AID-mediated
damage, but its role in Ig gene recombination
remains unclear. Here, we demonstrate that RPA
associates asymmetrically with resected ssDNA in
response to lesions created by AID, recombination-
activating genes (RAG), or other nucleases. Small
amounts of RPA are deposited at AID targets in
G1 in an ATM-dependent manner. In contrast,
recruitment in the S–G2/M phase is extensive, ATM
independent, and associated with Rad51 accumula-
tion. In the S–G2/M phase, RPA increases in nonho-
mologous-end-joining-deficient lymphocytes, where
there is more extensive DNA-end resection. Thus,
most RPA recruitment during class switch recombi-
nation represents salvage of unrepaired breaks by
phase of the cell cycle.
during the S–G2/M
Antigen receptor genes are assembled from variable (V), diver-
sity (D), and joining (J) gene segments by the V(D)J recombinase
Rag1 and Rag2 (Fugmann et al., 2000). This process requires
generation of DNA DSBs in lymphocyte precursors in the G1
phase of the cell cycle. These targeted DNA double-strand
breaks (DSBs) are joined by nonhomologous end joining
(NHEJ) as revealed by a complete block in Ig and T cell receptor
(TCR) gene assembly in the absence of NHEJ (Jung et al., 2006;
Rooney et al., 2004).
Upon antigenic stimulation the Ig genes in B lymphocytes
undergo additional diversification by somatic hypermutation
recombination reaction between highly repetitive switch regions
that replaces the heavy chain constant domain Igm with one of
several downstream isotypes Igg, Igε, or Iga (Honjo et al.,
2002; Stavnezer et al., 2008). Similar to V(D)J recombination,
NHEJ plays a key role in the resolution of DSBs incurred during
CSR (Helmink and Sleckman, 2012).
Both SHM and CSR are initiated by activation-induced cyti-
dine deaminase (AID), an enzyme that converts cytidines to
uracils at Ig variable genes and switch (S) regions (Maul et al.,
2011). dU:dG mismatches are recognized by base excision
and mismatch repair proteins leading to formation of DNA nicks
and DSBs that are obligate intermediates in CSR (Stavnezer
et al., 2008). In addition to Ig loci, AID can also target a large
number of non-Ig genes (Liu et al., 2008; Pasqualucci et al.,
1998; Shen et al., 1998; Yamane et al., 2011), including onco-
genes that are frequently translocated to Ig in human and mouse
B cell tumors (Chiarle et al., 2011; Klein et al., 2011; Ku ¨ppers and
How AID is targeted to Ig and non-Ig loci is still unknown, but
transcriptional pausing has been implicated (Peters and Storb,
1996; Rajagopal et al., 2009; Wang et al., 2009). In support of
this idea, the RNA exosome and RNA polymerase II stalling
factor Spt5 appear to be required for AID to access its target
genes (Basu et al., 2011; Pavri et al., 2010). Another potential
AID cofactor, the single-stranded DNA (ssDNA) binding
138 Cell Reports 3, 138–147, January 31, 2013 ª2013 The Authors
screen for activities that enhance AID hypermutation of in-vitro-
transcribed substrates (Chaudhuri et al., 2004). Consistent with
this idea, RPA associates with Ig and non-Ig AID target genes
(Vuong et al., 2009; Yamane et al., 2011) in a manner that is
directly proportional to the extent of AID activity (Hakim et al.,
2012). However, the precise role of RPA in CSR remains
In eukaryotic cells, RPA forms a complex with ssDNA that is
essential for DNA replication, telomere maintenance, DNA
recombination, DNA repair, and DNA damage checkpoint acti-
vation (Oakley and Patrick, 2010; Wold, 1997). There are at least
three ways whereby RPA might impact CSR. First, RPA could
help stabilize AID’s ssDNA targets during Ig gene transcription
base-excision repair (DeMott et al., 1998; Ranalli et al., 2002) or
mismatch repair (Genschel and Modrich, 2003; Lin et al., 1998),
which play critical roles in the processing of Ig gene deamination
(Stavnezer et al., 2008). Third, RPA might associate with and
stabilize resected ssDNA that cannot easily be repaired by clas-
sical NHEJ, thereby facilitating salvage by homology-mediated
repair pathways (Bothmer et al., 2010; Hasham et al., 2010;
Zhang et al., 2010).
RPA Accumulates in Response to DNA Breaks
Deletion of 53BP1 markedly increases RPA recruitment to Ig
genes, particularly in the presence of the IgkAID transgene (Fig-
ure S1; Hakim et al., 2012). To clarify the nature of RPA recruit-
ment at AID targets, we monitored RPA by chromatin immuno-
TCRα locus (chr14:52,972,107-54,890,656)
Figure1. RPARecruitmenttoAID,RAG, and
(A) RPA occupancy at the Igh locus of 53BP1?/?
(lane I), H2AX?/?(lane II), and H2AX?/?UNG?/?
Msh2?/?(lane III) B cells activated ex vivo in the
presence of LPS+IL-4. Deep-sequencing read
densities were normalized to adjust for library size
and bin width (reads per million per kb; RPKM).
Numbers in parentheses represent average read
density in RPKM for the displayed genomic
(B) TCRa locus showing recruitment of RPA in
53BP1?/?(lane I) and 53BP1+/+(lane III) control
thymocytes. Rag2 occupancy is also included in
(C) Kinetics of RPA recruitment at an I-SceI site
engineered at Myc intron 1. Activated B cells were
transduced withretroviruses expressing ER-I-SceI
and cells were harvested at 0, 0.5, 3, or 24 hr after
See also Figure S1.
precipitation (ChIP)-Seq in the absence
of H2AX, a factor that like 53BP1 limits
DNA-end resection during intrachromo-
somal recombination (Bothmer et al.,
2010; Helmink et al., 2011). B cells were
stimulated with lipopolysaccharide (LPS)
and interleukin 4 (IL-4) to promote CSR from Igm to Igg1 and
Igε. Similar to 53BP1?/?B cells, we found broad RPA islands
centered at recombining Sm, Sg1, and to a lesser extent Sε (Fig-
ure 1A, lanes I and II). Thus, absence of H2AX enhances RPA
deposition at Igh.
To ascertain whether RPA recruitment requires DSBs, as
opposed to AID recruitment, weperformed thesame experiment
in H2AX?/?UNG?/?Msh2?/?B cells. In this genetic background,
the resulting uracils are not processed to produce DSBs (Maul
et al., 2011; Rada et al., 2004; Xue et al., 2006). Notably, RPA
signals fell to background levels in the absence of DSBs (Fig-
ure 1A, lane III). This finding is consistent with the notion that
RPA deposition requires the formation of DSBs, but is indepen-
dent of DNA deamination and AID targeting.
To determine whether RPA is also associated with other DSBs
produced in G1, we examined CD4+CD8+double-positive
thymocytes, which actively undergo recombination-activating
gene (RAG)-mediated TCR-a recombination. Consistent with
extensive nucleolytic processing of coding ends in 53BP1?/?
T cells (Difilippantonio et al., 2008), we found prominent RPA
signals in Ja (Figure 1B, lane I), a profile that is reminiscent of
RAG2 recruitment to this locus (Figure 1B, lane II; Ji et al.,
2010). In contrast to wild-type B cells, which accumulate RPA
at the Ig locus during CSR, RPA accumulation in T cells required
53BP1 deletion, because ChIP signals were indistinguishable
from background in wild-type thymocytes (Figure 1B, lane III).
To verify that DSBs are sufficient to stimulate RPA accumula-
tion and to examine the kinetics of recruitment, we induced DNA
breaks in 53BP1?/?AID?/?MycI/IB cells by expression of an
estrogen inducible I-SceI meganuclease (ER-I-SceI) (Robbiani
Cell Reports 3, 138–147, January 31, 2013 ª2013 The Authors 139
et al., 2008). RPA recruitment became detectable 3 hr following
Taken together, the data demonstrate that in 53BP1?/?lympho-
cytes RPA accumulates rapidly in response to physiological
DSBs introduced in G1 (RAGs, AID) as well as nuclease-induced
RPA Associates with Resected ssDNA
During DNA repair RPA associates with ssDNA created by end
resection (Symington and Gautier, 2011). Resected DNA is typi-
cally asymmetric and immunoprecipitated RPA associated with
such DNA structures would be expected to reflect that asymme-
try. Indeed, when RPA ChIP-seq libraries from 53BP1?/?IgkAID
B cells were resolved into upper (+) and lower (?) DNA strands
RPA was asymmetrically distributed around AID target sites.
As exemplified by Grap, a non-Ig AID target gene (Klein et al.,
2011; Yamane et al., 2011), RPA was enriched on the + strand
upstream, and on the ? strand downstream of intron 1 (Fig-
ure 2A). This distribution is consistent with 50–30DNA-end resec-
tion (Figure 2A, schematics). Directionality in RPA recruitment
was also observed at Igh and other AID target genes in
53BP1?/?B cells (Figure 2B and not shown). Similar results
were also obtained with MycIcells transduced with I-SceI (Fig-
ure S2A). In contrast to RPA, PolII ChIP-seq libraries did not
display any obvious strand biases at Grap or other AID target
genes (Figure 2B and not shown).
To confirm that RPA binds to resected DNA at AID target
genes, we incubated RPA and control PolII immunoprecipitates
with Escherichia coli single-strand exonuclease ExoI, which
digests ssDNA from 30-50before library preparation. While PolII
signals were largely unchanged, RPA enrichment at Grap or
other AID targets could not be detected after nuclease treatment
Figure 2. RPA Interacts with Resected ssDNA Downstream of AID or I-PpoI Endonuclease
(A) RPA association with + and ? DNA strands at the Grap gene locus from IgkAID-53BP1?/?-activated B cells. RPKM values for the specified window
(chr11:61,401,372-61,543,038) are provided in parenthesis.
(B) RPA bound to Igh locus. As in (A), ChIP-seq signals were resolved into upper and lower strands. Technical replicates were treated with E. coli ExoI or RecJ
exonucleases nuclease prior to deep-sequencing library preparation.
(C) PolII binding to + and ? strands at the Grap locus.
(D) Technical IP replicates from (A) and (C) samples incubated in the presence of ExoI.
(E) Composite diagram showing RPA profiles at 19 I-PpoI mouse genomic sites in 53BP1?/?MEFs transduced with retroviruses expressing the I-PpoI homing
See also Figure S2.
140 Cell Reports 3, 138–147, January 31, 2013 ª2013 The Authors
(Figures 2C and 2D; Figure S2B). Although to a lesser extent,
treatment of IP DNA with RecJ, a 50–30exonuclease, also dimin-
ished RPA signals substantially (Figures 2B and 2D; Figure S2B).
This observation is consistent with the notion that resected,
200bpsonicated DNAissusceptible toboth50and30nucleolytic
attack (Figure S2C). On the basis of these findings, we conclude
that RPA associates with resected DNA in response to AID or
nuclease-mediated DNA damage.
In yeast, DNA-end resection can extend up to 10 kb in the
absence of a homologous donor or Rad51 (Bishop et al., 1992;
of DNA-endresection in mammalian cellshowever areunknown.
Because AID-mediated damage is not site specific but occurs
over a relatively large genomic domain (?1–3 kb; Klein et al.,
2011; Yamane et al., 2011), we monitored DNA-end resection
in 53BP1?/?MEFs transduced with the I-PpoI endonuclease,
which recognizes 19 sites in the C57BL/6 genome (San Filippo
et al., 2008). RPA recruitment to all 19 I-PpoI sites was strand
biased, centered on the DSB, and extended over an area of
slightly more than 20 kb (Figure 2E). Analogous results were
also obtained at Myc upon I-SceI expression in MycIB cells (Fig-
ure S2A). We conclude that in mammalian cells deficient in
53BP1, resection occurs for up to 10–15 kb on either side of
Rad51 Recruitment to AID-Mediated Breaks
RPA has been studied primarily in the context of HR where it
recruits Rad51, a recombinase that forms the presynaptic nucle-
oprotein filament required for strand invasion (Ogawa et al.,
1993; Sung and Robberson, 1995). To determine whether
Rad51 also associates with resected DNA damaged by AID,
we performed Rad51 ChIP-seq on 53BP1?/?IgkAID B cells. We
found extensive Rad51 recruitment at Igh (Figure 3A). Analogous
to RPA, the Rad51 ChIP signal was centered at recombining
switch domains and displayed a marked DNA strand bias
(Figure 3A). In addition, Rad51 was also found at AID off-target
genes, such as Mir155 and Cd83 (Figure 3B). Additional exam-
ples are provided in Figure S3. At these sites, the domain
separating the strand-specific Rad51 islands overlapped with
hotspots of AID-induced chromosomal translocations (Fig-
ure 3B), as determined by TC-Seq (Klein et al., 2011). Moreover,
the relative amount of Rad51 deposition per off-target gene
(TSS ± 2 kb) was directly proportional to its translocation
frequency (Spearman r = 0.65, Figure 3C). This result is consis-
tent with the notion that, as described for RPA (Hakim et al.,
2012), the extent of Rad51 occupancy is a function of AID
activity. In contrast, genes not associated with RPA displayed
background levels of Rad51 (Figure 3D and not shown). We
conclude that in the absence of 53BP1, RPA and Rad51 asso-
ciate with sites of AID-mediated damage at Ig and non-Ig genes.
RPA Recruitment Occurs Mostly in S–G2/M
Recruitment of RPA and Rad51 to resected DNA might occur in
G1 during NHEJ repair of V(D)J or CSR DSBs. Alternatively, RPA
could be recruited in S and G2/M phases of the cell cycle as
part of a salvage mechanism for unrepaired DSBs. To address
this question, we purified H2AX?/?lymphocytes in the G1, S,
and G2/M phases of the cell cycle and performed RPA
ChIP-seq. Consistent with limited DNA-end resection during
NHEJ (Bothmer et al., 2010; Helmink et al., 2011), G1 cells dis-
played localized RPA deposition at the Igh locus (Figure 4A) in
a manner that overlapped with sites of AID activity (Hakim
et al., 2012; Klein et al., 2011). In contrast, lymphocytes from S
and G2/M phases recruited far greater amounts of RPA (Fig-
ure 4A). Analogous resultswere obtained using53BP1?/?Bcells
(see below). Thus, RPA recruitment occurs in both G1 andS–G2/
M, with the majority of the signal accumulating in cells that prog-
ress to S–G2/M.
Figure 3. Rad51 Recruitment to AID On- and Off-Targets
IgkAID-53BP1?/?-activated B cells.
(B) Rad51 (blue), RPA (red), and translocation profiles at Mir155 and Cd83
(C) Chromosomal translocations (TSS ± 2 kb) involving Igh or Myc at Rad51-
recruiting genes. Chromosomes 12 and 15 carrying the I-SceI sites were
excluded from the analysis. Correlation between the two data sets is calcu-
lated using Spearman’s r.
(D) Rad51 levels (TSS ± 2 kb) at RPA+ and RPA? genes in IgkAID-53BP1?/?
See also Figure S3.
Cell Reports 3, 138–147, January 31, 2013 ª2013 The Authors 141
Our results suggested that in end-joining-deficient cells,
a significant fraction of CSR lesions produced in G1 persist
into S and G2/M, where they are processed for HR. To explore
this possibility, we monitored Igh breaks in cycling cells by
ChIP for thephosphorylated histone H2AX (gH2AX),which accu-
mulates at sites of DSBs (Rogakou et al., 1999). In wild-type B
cells in G1, gH2AX was primarily associated with Igh (1.40 reads
per million per kb [RPKM], Figure 4B, lane I), and these signals
decreased in S phase cells to levels found in H2AX?/?controls
(Figure 4B, lanes II and III). These results support the idea that
most CSR breaks are resolved before DNA replication (Hasham
et al., 2012; Petersen et al., 2001; Schrader et al., 2007; Shar-
been et al., 2012). In contrast, 53BP1?/?B cells showed similar
levels of gH2AX accumulation in G1 and in the S phase (Fig-
ure 4B, lane V), indicating that in end-joining-deficient cells
a significant fraction of CSR breaks persist beyond G1, even in
the presence of intact ATM and p53 (Calle ´n et al., 2007). These
data indicate that unrepaired AID-mediated DNA damage does
not efficiently activate cell-cycle checkpoints in the absence of
53BP1 or H2AX.
As expected based on results with unsorted samples (Fig-
ure S2A), RPA displayed strand biases at Igh in all cell-cycle
stages (Figure 4C; data not shown). In contrast, gH2AX signals
were not asymmetric around S domains (Figure 4C), indicating
that this chromatin mark, unlike RPA, is mostly associated with
unresected double-strand lesions. The persistence of gH2AX
signals in G2/M-phased cells is consistent with the presence of
frequent chromosome 12 breaks in NHEJ-deficient metaphases
(Bothmer et al., 2011; Bunting et al., 2010; Gostissa et al., 2011).
ATM Is Required for Resection in G1 but Not S–G2/M
ATM is required for DNA-end resection during CSR, V(D)J
recombination, and in cells treated with clastogenic agents
(Bothmer et al., 2010, 2011; Helmink et al., 2011; Jazayeri
et al., 2006). To examine the role of ATM in RPA recruitment
during CSR, we treated LPS+IL-4-activated B cells with the
small molecule ATM inhibitor KU-55933 (ATMi) and performed
RPA ChIP-seq on purified G1 and S–G2/M phase cells. ATMi-
treated G1 phase cells showed only background RPA signals
at non-Ig AID targets in the absence of 53BP1. In addition,
RPA was 3-fold lower at Igh in ATMi-treated cells relative to
controls (p = 0.02; Figure 5B; n = 3). In contrast, activated B cells
et al., 2012) did not show this effect (Figure 5B), which is con-
sistent with the observation that ATR is activated only after
significant resection has occurred (Brown and Baltimore, 2003;
Pellicioli et al., 2001; Shiotani and Zou, 2009; Zou and Elledge,
2003). Furthermore, ATR inhibitor treatment did not affect the
extent of resection in S–G2/M (Morin et al., 2008).
To confirm a role for ATM in initiating resection in G1, we
examined 53BP1?/?and 53BP1?/?ATM?/?thymocytes. The
analysis showed a substantial reduction in RPA deposition at
the TCRa locus in the absence of ATM (Figure 5C). Consistent
with ATMi results, RPA signals at Igh in unfractionated B cells
undergoing CSR from the same mice were not affected (Fig-
ure 5D). Taken together, these findings demonstrate that ATM
is required to promote initiation of RPA recruitment to resected
DNA in G1- but is not required for its recruitment in S–G2/M-
We have shown that in NHEJ-deficient cells (H2AX?/?or
53BP1?/?), a fraction of DNA breaks that are normally repaired
in G1 become substrates for homologous-mediated repair in
S/G2/M. To investigate whether this also occurs under physio-
logical conditions, we measured Rad51 and RPA occupancy
in activated wild-type B cells undergoing CSR. We found
strand-biased recruitment of Rad51 and RPA at Igm and Igg1
(Figure 5E), consistent with 50–30resection activity in wild-type
cells. Resection required AID activity because RPA signals
wereincreased inthepresence ofIgkAID andwere indistinguish-
able from background in AID?/?lymphocytes (Figure 5E). As ex-
pected, the extent of resection in wild-type cells was reduced
relative to H2AX?/?or 53BP1?/?(compare RPKM values to
Figures 2B and 5D, for instance). We thus conclude that recom-
bining Ig genes are resected under physiological conditions,
arguing that nonhomologous and homologous repair pathways
contribute to repair during CSR.
Our results show that RPA is recruited to resected DNA DSBs
produced by AID, RAGs, and site-specific endonucleases.
II S (WT)
III G1 (H2AX-/-)
I G1 (WT)
IV G1 (53BP1-/-)
V S (53BP1-/-)
11chr12: 14,438,366- 14,731,719
Figure 4. RPA and gH2AX Recruitment during Cell Cycle
(A) RPA accumulation in H2AX?/?-activated B cells at the Igh locus during the
cell cycle. Samples were stained with Hoechst dye and sorted into G1, S, and
G2/M-phased cells. RPKM values for the specified genomic windows are
provided in parentheses.
(B) Extent of H2AX phosphorylation (gH2AX) at the Igh locus in wild-type (G1,
lane I; S, lane II), H2AX?/?(G1, lane III), or 53BP1?/?(G1, lane IV; S, lane V)-
activated B cells.
(C) RPA (G1, G2/M) and gH2AX (G2/M) deposition at Igh in cells from H2AX?/?
or 53BP1?/?respectively. ChIP-seq data were split into + and – strands.
142 Cell Reports 3, 138–147, January 31, 2013 ª2013 The Authors
RPA accumulation begins within 3 hr in the G1 phase of the cell
cycle and unrepaired lesions persist into the S and G2/M phases
where HR or other salvage repair mechanisms resolve DSBs.
While our findings do not exclude the possibility that RPA can
enhance AID activity (Chaudhuri et al., 2004), we can conclude
that RPA recruitment is not essential for Ig gene deamination
RPA recruitment to DNA damage is asymmetric around DSBs,
displaying a strongbias for the +DNAstrand 50,and the ? strand
30of the break. This profile is best explained by the 50–30direc-
tionality of DNA-end resection, which we find spreads 10–15
described in yeast (Bishop et al., 1992; Sugawara et al., 1995;
Zhu et al., 2008). Consistent with this idea, exonuclease treat-
ment of immunoprecipitated DNA, a procedure that specifically
removes ssDNA 30tracks, ablates RPA ChIP signals. In addition,
Rad51, which is required for strand invasion during HR, mirrors
RPA deposition. The striking overlap between RPA and Rad51
profiles is consistent with the idea that RPA accumulation on re-
sected DNAin S–G2/M facilitates HR (Newet al., 1998, Figure 6).
DNA-end resection has been primarily studied in yeast, where
trimming phase, and the Exo1 and the RecQ helicase Sgs1
extend the resected tracks (Mimitou and Symington, 2008; Sy-
mington and Gautier, 2011; Zhu et al., 2008). The mechanistic
details of end resection in mammalian cells are less clear, but
both CtIP and ATM appear to play a role. During V(D)J recombi-
RPA (RPKM) G1/S-G2M
P = 0.02
Figure 5. ATM Is Required for G1 but Not
(A) RPA accumulation at Pim1 and IL4Ra loci from
53BP1?/?-activated B cells that were either
treated (lower two panels) or not treated (upper
panels) with the ATM inhibitor KU-55933. Samples
were sorted into G1 or S/G2/M-phased cells using
the Hoechst dye 33342. Numbers in parentheses
represent RPKM values within the specified
(B) RPA accumulation at Igm and Igg1 loci in WT,
ATMi-, or ATRi-treated B cells. Values represent
the RPKM ratio between G1 and S–G2/M-phased
cells. Error bars represent the SD of four biological
replicates (two for Igm and two for Igg1) for each
experimental condition with the exception of ATRi.
p value was calculated using the unpaired t test.
(C and D) RPA recruitment to the TCRa in thymo-
cytes (C) or Igh in activated B cells (D) from
53BP1?/?(upper) or 53BP1?/?ATM?/?(lower)
(E) RPA and Rad51 recruitment to activated Bcells
with an intact NHEJ: IgkAID transgenics, AID+/+,
nation, depletion of CtIP in H2AX?/?cells
protects DSBs from end resection (Hel-
mink et al., 2011). In like manner, inhibi-
tion of ATM in 53BP1?/?B cells protects
CSR breaks from degradation (Bothmer
et al., 2010). Our results confirm a role
for ATM in the resection of V(D)J and
CSR lesions, but notably, only in the G1 phase of the cell cycle.
Thus, an ATM-independent pathway controls resection at post-
The accumulation of RPA at resected AID target genes in the
S–G2/M phases of the cell cycle suggest that some AID-induced
DSBs are carried over from G1 to S where they would be prefer-
entially repaired by HR. Consistent with this model, B cells
deficient in the HR factor XRCC2 accumulate unrepaired AID-
mediated breaks at postreplicative stages of the cell cycle (Ha-
sham et al., 2010, 2012). However, the mechanism that allows
these lesions to persist beyond G1 was not resolved. ATM is
an essential mediator of the G1-S phase checkpoint, and when
it is deleted DSBs persist through cell-cycle phase transitions
leading to chromosome breaks and genome instability (Brede-
meyer et al., 2006; Calle ´n et al., 2007; Derheimer and Kastan,
2010). While it is conceivable that the relative low number of
AID-mediated DSBs per cell might be insufficient to activate
the checkpoint, this scenario seems unlikely because ATM can
be activated by only a few breaks (Bakkenist and Kastan,
2003; Chen et al., 2000; Petersen et al., 2001). Moreover, AID
activity engages ATM as indicated by formation of gH2AX foci
on recombining Igh loci (Petersen et al., 2001). Cells that resolve
thesebreaksbyNHEJ extinguish ATMand progress. Incontrast,
DSBs that fail to resolve would be expected to undergo further
processing, including end resection and RPA recruitment, which
can suppress ATM signaling and abort the G1-S checkpoint
(Calle ´n et al., 2007; Shiotani and Zou, 2009). Thus, RPA
Cell Reports 3, 138–147, January 31, 2013 ª2013 The Authors 143
accumulation in G1 may help avert the G1-S checkpoint and
facilitate homology-mediated repair by enabling progression of
cells carrying unrepaired breaks from G1 into the S phase.
What is the role of homology-mediated DNA repair during
CSR? AID initiates CSR by introducing multiple DSBs in highly
repetitive switch regions, which vary in length from 1 to ?10 kb
(Honjo et al., 2002). NHEJ repair of paired DSBs in heterologous
switch regions results in productive CSR. However, ?20% of the
alternative form of nonhomologous end joining (A-NHEJ) that
promotes intraswitch deletions (Bottaro et al., 1998; Dudley
et al., 2002; Gu et al., 1993; Hummel et al., 1987; Reina-San-
Martin et al., 2003; Winter et al., 1987). We propose that this
salvage mechanism is mediated by S region microhomology
uncovered by DNA-end resection (Bothmer et al., 2010, 2011)
and RPA recruitment (Figure 6). The presence of localized RPA
at Igm and Igg1 in the G1 phase of the cell cycle is consistent
with this idea. Whether A-NHEJ is also active at Igh in S–G2/M
remains to be determined. Our results also imply that DNA
breaks notprocessed bythecanonical or alternative NHEJ path-
ways would be further resected in S-G2/M and repaired by HR
using the second, intact Igh allele (Figure 6). The physiologic
advantage of engaging error-free HR during CSR is that switch
regions thus repaired would be available for subsequent recom-
of chromosomal translocations.
IgkAID (Robbiani et al., 2009); UNG?/?(Endres et al., 2004); ATM?/?(Barlow
et al., 1996); AID?/?(Muramatsu et al., 2000), 53BP1?/?(Ward et al., 2003),
MycI(Robbiani et al., 2008); Msh2?/?(Reitmair et al., 1995); and H2AX?/?
(Bassing et al., 2003) mice were previously described. All experiments were
in accordance with protocols approved by the NIAMS and Rockefeller Institu-
tional Animal Care and Use Committee.
Ex vivo cultured B cells or thymocytes were fixed with 1% formaldehyde
(Sigma) for 10 min at 37?C. The fixation was quenched by addition of glycine
(Sigma) at a final concentration of 125 mM. Twenty million fixed cells were
washed with PBS and then resuspended in 1 ml of RIPA buffer (10 mM Tris
[pH 7.6], 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton
X-100, 13 Complete Mini EDTA free proteinase inhibitor [Roche]) or stored
at ?80?C until further processing. The sonication was performed on S2 son-
icator (Covaris) at duty cycle 20%, intensity 5, cycle/burst 200 for 30 min. Ten
micrograms of anti-RPA32 (EMD, NA19L), anti-RAD51 (Santa Cruz, H-92), or
anti-gH2AX (Epitomics, 2212-1) was incubated with 40 ml of Dynabeads
Protein A for 40 min at room temperature. The antibody-bound beads were
added to 500 ml of sonicated chromatin, incubated at 4?C overnight, and
washed twice with RIPA buffer, twice with RIPA buffer containing 0.3M
NaCl, twice with LiCl buffer (0.25 M LiCl, 0.5% Igepal-630, 0.5% sodium de-
oxycholate), once with TE (pH 8) plus 0.2% Triton X-100, and once with TE
(pH 8). Crosslinking was reversed by incubating the beads at 65?C for 4 hr
with 0.3% SDS and 1 mg/ml Proteinase K. ChIP DNA was purified by
phenol-chloroform extraction followed by ethanol precipitation. The DNA
was subsequently blunt-ended with End-It DNA end repair kit (Epicenter)
and A-tailed with Taq DNA polymerase (Invitrogen) in the presence of
200mM of dATP for 40 min at 70?C. The sample was purified by phenol-
chloroform extraction after each reaction. Illumina compatible adaptors (Illu-
mina or Bioo Scientific) were then ligated with T4 DNA ligase (Enzymatics),
and the reaction was purified once with AMpure XP magnetic beads (Beck-
man Coulter). Samples were PCR amplified for 18 cycles with KAPA HiFi
DNA polymerase mix (KAPA Biosystems). The amplicon was run on a 2%
agarose gel and size-selected at 200–300 bp. Thirty-six or 50 bp of
sequencing data were acquired on GAII or HiSeq2000 (Illumina). For nuclease
treatment, ChIP DNA was incubated for 2 hr with 20 units of E. coli ExoI (New
England Biolabs) at 37?C or 30 units of RecJf nuclease (New England Biolabs)
at 37?C for 2 hr prior to the blunt-end ligation step. How ssDNA is amplified
and detected during the Illumina library protocol has been recently addressed
by two independent studies (Croucher et al., 2009; Khil et al., 2012). In brief,
ssDNA is rendered double-stranded by intermolecular or intramolecular
(hairpin) priming, facilitated by DNA microhomologies (Croucher et al.,
2009; Khil et al., 2012). Table S1 contains a list of all ChIP-seq experi-
ments as they appear in the manuscript figures. Biological replicates or com-
parable experiments that further support the results presented in each panel
are also provided.
Cell Culture and Retroviral Infection
For I-SceI assays, CD43-resting B cells were isolated from spleens, stimu-
lated, and infected as previously described (Bothmer et al., 2011). The
pMX-IRES-GFP based retrovirus encoding I-SceI was previously described
(Robbiani et al., 2008). The new pMX-ER-I-SceI was generated by fusing in-
frame the optimized ligand-binding domain of the human estrogen receptor
(ERT2) with I-SceI. For I-PpoI assays, immortalized 53BP1?/?mouse embry-
onic fibroblasts (MEFs) were transduced with the pMY retroviral vector encod-
ing BFP-I-PpoI fusion gene. Following spin-infection MEFs were reseeded
until they reached ?70% confluency, at which time cells were trypsinized for
5seconds at37?C,andtrypsinwasquenched withcompletemediacontaining
1.25% formaldehyde for 10 seconds. Following incubation a final concentra-
tion of 125 mM glycine was added to stop crosslinking.
Flow Cytometry and Cell Sorting
Dickinson) or Moflo (Beckman Coulter). For cell-cycle sorting, B cells were
cultured for 72 hr with LPS, IL-4, and anti-Cd180 antibody and incubated for
an additional for 45 min in the presence of 10 mg/ml of Hoechst 33342 (Invitro-
gen). Cells were then fixed the same conditions used for ChIP-seq sample
preparation. Samples were the resuspended in HBSS buffer containing
Figure 6. DNA-End Resection during CSR
Model showing how AID-mediated breaks at Igh are either processed by C-
NHEJ proteins into efficient switch recombination, or are resected by ATM-
dependent A-NHEJ in G1 or by the HR in S–G2/M, leading to RPA recruitment
and repair. Based on previous work (Shiotani and Zou, 2009), extensive
resection in connection with HR is expected to inhibit ATM activity and
potentially reduce the formation of chromosomal translocations via A-NHEJ.
144 Cell Reports 3, 138–147, January 31, 2013 ª2013 The Authors
machines (BD Biosciences) using 355 or 375 nm lasers, respectively.
Deep-sequencing data are available at GEO under accession number
Supplemental Information includes three figures and one table and can be
This is an open-access article distributed under the terms of the Creative
Commons Attribution-NonCommercial-No Derivative Works License, which
permits non-commercial use, distribution, and reproduction in any medium,
provided the original author and source are credited.
We thank all members of the Casellas and Nussenzweig labs for helpful
discussions, Matthias F. Muellenbeck, Jim Simone, Jeff Lay, and Gustavo Gu-
ral program of NIAMS at the NIH. This study utilized the high-performance
computational capabilities of the Helix/Biowulf Systems at the National Insti-
tutes of Health, Bethesda, MD (http://helix.nih.gov).
Received: September 21, 2012
Revised: November 14, 2012
Accepted: December 12, 2012
Published: January 3, 2013
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