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of BRCA1 is important for the survival of cells after
IR. 51 Among its many functions, BRCA1 associates
with BARD1 to form an E3 ubiquitin ligase. BRCA1
mutations that disrupt the E3 ligase activity render cells
sensitive to IR. 52 The E3 activity of BRCA1 is stimulated
on chromatin after IR in an ATM- and ATR-dependent
manner. It has been shown that the interaction between
BRCA1 and the E2 ubiquitin conjugating enzyme
UbcH5c is dependent upon both ATM and ATR. 53
BRCA1 associates with CtIP during G2 phase of the
cell cycle, and it ubiquitinates CtIP on chromatin. 54
The ubiquitination of CtIP does not result in its degra-
dation, but is required for the normal G2/M checkpoint
response. Whether and how the E3 ligase activity of
BRCA1 contributes
with ATR at sites of DSBs and stalled replication
forks. 62,63 The binding of Slx4 and Dpb11 may provide
a means to recruit the Slx4-containing complex to sites
of DNA damage. Although whether human SLX4 is
recruited to sites of HR in an ATM/ATR-dependent
manner remains to be tested, the data on yeast Slx4 raise
the interesting possibility that ATM and/or ATR are
important for the assembly of HR complex after strand
invasion.
Chromatin-Mediated Recruitment of Repair
Proteins
When DSBs are generated in chromosomes, ATM not
only phosphorylates DNA repair proteins recruited to
DNA ends but also the histone variant H2AX in nucle-
osomes adjacent to DSBs. 64 DNA-PK, another protein
kinase activated by DSBs, also contributes to H2AX
phosphorylation. 65 The phosphorylation of H2AX at
Ser 139 by ATM and DNA-PK is directly recognized
by MDC1. 66 MDC1 in turn binds to autophosphory-
lated ATM and the MRN complex, allowing ATM to
accumulate on chromatin and phosphorylate more
H2AX in adjacent nucleosomes. 67 This positive feed-
back loop enables ATM to propagate H2AX phosphor-
ylation in large chromosomal regions flanking DSBs
( Figure 7.3 ).
Phosphorylated H2AX (known as g -H2AX) around
DSBs facilitates the recruitment of a number of DNA
repair proteins and chromatin modulating factors. In
particular, through MDC1, g -H2AX recruits the RNF8
E3 ubiquitin ligase. 68 e 71 RNF8 catalyzes the formation
of ubiquitin chains on histones H2A and H2AX around
DSBs ( Figure 7.3 ). The ubiquitin chains generated
by RNF8 subsequently recruits two additional E3
ligases, RNF168 and HERC2, which help to extend
the ubiquitin chains through Lys63-mediated linkage
( Figure 7.3 ). 72 e 74
The Lys 63-linked ubiquitin chains around DSBs play
an important role in the accumulation of BRCA1 at these
sites. BRCA1 exists in at least three distinct complexes in
cells. Through the BRCT (BRCA1 C terminal) domains,
BRCA1 associates with either RAP80, BACH1, or CtIP,
which define the BRCA1-A, B, C complexes, respec-
tively. 75 The RAP80 in the BRCA1-A complex directly
recognizes the Lys 63-linked ubiquitin chains via a ubiq-
uitin-interacting motif (UIM), thereby localizing the
BRCA1-A complex to DSBs ( Figure 7.3 ). 75 e 77 Additional
components of the BRCA1-A complex include ABRA1,
BRE1, BRCC36, and NBA1. 78 e 82 Interestingly, several
of the components of the BRCA1-A complex display
structural similarities to the components of the protea-
some lid complex. 82 The exact role of BRCA1-A complex
in HR still needs to be determined. In addition to
BRCA1, the Lys 63-linked ubiquitin chains also recruits
the RAD18 ubiquitin ligase. 83
to HR still
requires
further
investigations.
HRR not only occurs at DSBs but also at stressed or
stalled DNA replication forks (see HRR at Stalled or
Collapsed DNA Replication Forks, below, for details).
Unlike DSBs, stalled replication forks primarily activate
ATR but not ATM. 55 In response to the DNA replication
inhibitor hydroxyurea (HU), RAD51 is phosphorylated
by Chk1, an effector kinase of ATR. 56 The phosphoryla-
tion of RAD51 by Chk1 is important for the accumula-
tion of RAD51 at HU-induced nuclear foci, which
presumably promotes HR at stalled replication forks.
RPA2, the middle subunit of the trimeric RPA
complex, is phosphorylated at multiple sites in an
ATM- and ATR-dependent manner after IR or HU treat-
ment. The phosphorylation of RPA2 is required for the
formation of RAD51 foci only in response to HU but
not IR, suggesting that it may be specifically involved
in HRR at stalled replication forks. 57 Interestingly,
RPA2 also needs to be dephosphorylated by the PP2A
phosphatase to support normal recovery from the HU-
induced replication stress. 58 In response to IR, the
dephosphorylation of RPA2 by the PP4 phosphatase is
required for the formation of RAD51 foci and efficient
HR. 59 These findings suggest that RPA2 phosphoryla-
tion can positively and negatively regulate the formation
of RAD51 filament in a context-specific manner.
Recruitment of Nucleases
Recent studies suggested that SLX4 is an important
scaffold for a large complex of DNA repair proteins. 31 e 34
Among the repair proteins associated with human SLX4
are the SLX1 and MUS81 e EME1 endonucleases, which
are important for the processing of HR intermediates
after strand invasion. In human cells, SLX4 is phosphor-
ylated in an ATM/ATR-dependent manner in response
to DSBs. 60 The yeast Slx4 is also phosphorylated by the
ATR counterpart Mec1 after DNA damage. The phos-
phorylation of yeast Slx4 enables it to bind Dpb11, the
yeast homolog of human TopBP1. 61 TopBP1 is an acti-
vator of the ATR e ATRIP kinase, and it colocalizes
It has been shown that
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