Biology Reference
In-Depth Information
generates non-crossover repair products (
Figure 7.2
).
Alternatively, extension of the invading strand leads to
migration of the D loop, allowing it to capture the
second end of DSBs and to form two HJs. As discussed
in the following section, HJs can be resolved in different
ways, generating either crossover or non-crossover
products.
Several proteins have been implicated in strand inva-
sion and HJ formation in human cells. Human RAD54
and RAD54B are members of the SWI/SNF2 family
ATPases. Knockout mouse models of RAD54 and
RAD54B suggest that these two proteins function redun-
dantly during HRR.
23
The sole Rad54 protein in yeast
displays dsDNA-dependent ATPase, DNA translocase,
DNA supercoiling and chromatin remodeling activities
in vitro
, and it promotes the formation of D loop by
Rad51 filament.
24
Rad54 is also able to remove Rad51
from dsDNA in an ATP hydrolysis-dependent manner,
which is important for the late stages of HR. Further-
more, humans have five RAD51 paralogues (RAD51B,
RAD51C, RAD51D, XRCC2, and XRCC3).
25,26
These
RAD51 paralogues form heterodimers, tetrameric
complexes, and interact with the RAD51 filament. There
is evidence that RAD51 paralogs facilitate the assembly
and/or maintenance of RAD51 filament, and enhance
the homologous DNA pairing activity of RAD51.
these nucleases function in a coordinated way during
HRR or in different DNA repair pathways.
The Role of ATM and ATR Checkpoint
Kinases in HRR
Both the ATM and ATR checkpoint kinases are acti-
vated by DSBs in human cells. The activation of these
kinases regulates and coordinates multiple cellular
processes, such as cell cycle progression, DNA
synthesis, transcription response, and apoptosis. Impor-
tantly, both ATM and ATR play critical roles in DNA
repair at sites of DSBs.
The MRN complex is a direct sensor of DSBs in cells.
NBS1 in the MRN complex directly interacts with ATM
and recruits ATM to DSBs. In a DNA end-dependent
manner, MRN stimulates the kinase activity of ATM,
enabling ATM to phosphorylate its substrates at
DSBs.
37
During the activation of ATM, ATM is autophos-
phorylated at multiple sites including Ser 1981
(
Figure 7.1
).
38-40
As described below, ATM is required
for the efficient resection of DNA ends. Once DNA
ends are progressively resected, the association of
MRN with DNA ends is gradually weakened, and the
ability of DNA ends to activate ATM attenuated.
41
At
the same time, the ssDNA generated by resection is
coated by RPA, which directly interacts with ATRIP,
the functional partner of ATR, and recruits the ATR-
ATRIP kinase complex.
41,42
Thus, the resection of DNA
ends promotes the consecutive activation of ATM and
ATR at DSBs (
Figure 7.1
). This regulatory mechanism
may allow ATM and ATR to function jointly and to carry
out distinct roles during HRR.
Dissolution of Holliday Junctions
Once HJs are formed, they can migrate on DNAwith
the help of translocases. RAD54, FANCM, and the RecQ
helicases (BLM, WRN, RECQL1 and RECQ5b) are able
to branch migrate HJs
in vitro
.
27
e
29
To complete HRR,
HJs have to be resolved. One mechanism to resolve
HJs is mediated by the BLM-TOPOIII-RMI1 complex.
30
This mechanism suppresses the generation of crossover
products (
Figure 7.2
). The second mechanism for HJ
dissolution is mediated by a group of endonucleases
(so called the Holliday junction resolvases). MUS81-
EME1, GEN1, and SLX1-SLX4 are able to resolve HJs
in vitro
.
31
e
36
Cleavage of HJs by MUS81-EME1 generates
asymmetric products that cannot be ligated, suggesting
that it may not be a classic HJ resolvase. It is speculated
that MUS81-EME1 cleaves fork-like structures in D
loops during HRR. On the other hand, both GEN1 and
SLX1-SLX4 cut HJs symmetrically and generate prod-
ucts that can be ligated. Given its symmetry, each HJ
can be cut by HJ resolvases in two different ways.
Depending on how double HJs are cut by SLX1-SLX4
and/or GEN1, this mechanism can give rise to either
crossover or non-crossover products (
Figure 7.2
). While
the putative HJ resolvases have been extensively charac-
terized
in vitro
, their functions
in vivo
are not fully
understood. Interestingly, in human cells, SLX4 not
only associates with SLX1 but also MUS81-EME1 and
another endonuclease ERCC1/XPF. It is possible that
Resection of DNA ends
ATM is required for the efficient resection of DNA
ends and the subsequent activation of ATR.
11,12,41
Several proteins involved in resection, including NBS1,
MRE11, CtIP, EXO1, and BLM, are phosphorylated by
ATM after DNA damage.
43
e
48
In vitro
studies using
Xen-
pous
egg extracts suggested that the recruitment of CtIP
to DSBs is dependent upon ATM, although the exact
mechanism of this event is still unclear.
49
In human cells,
the phosphorylation of EXO1 by ATM appears to be
important for efficient resection.
48
Overexpression of
CtIP and EXO1 accelerates resection in the presence of
ATM, but it does not bypass the requirement of ATM
for resection,
41
suggesting that ATM is important for
the functions of CtIP and EXO1 or it has additional
targets important for resection.
Formation of RAD51 Filament
In response to DSBs, BRCA1 is phosphorylated by both
ATM and ATR at mul t iple si tes .
50
The phosphorylation
