Mismatch repair protein MSH2 regulates translesion DNA synthesis following exposure of cells to UV radiation
Lingna Lv
2
Fengli Wang
1
Xiaolu Ma
2
Yeran Yang
2
Zhifeng Wang
2
Hongmei Liu
1
Xiaoling Li
1
Zhenbo Liu
2
Ting Zhang
2
Min Huang
2
Errol C. Friedberg
0
Tie-Shan Tang
1
Caixia Guo
2
0
Department of Pathology, University of Texas Southwestern Medical Center
,
Dallas, TX 75390, USA
1
State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences
,
Beijing 100101, China
2
Laboratory of Cancer Genomics and Individualized Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences
,
Beijing 100101, China
-
Translesion DNA synthesis (TLS) can use
specialized DNA polymerases to insert and/or
extend nucleotides across lesions, thereby limiting
stalled replication fork collapse and the potential for
cell death. Recent studies have shown that
monoubiquitinated proliferating cell nuclear
antigen (PCNA) plays an important role in
recruitment of Y-family TLS polymerases to stalled
replication forks after DNA damage treatment. To
explore the possible roles of other factors that
regulate the ultraviolet (UV)-induced assembly of
specialized DNA polymerases at arrested replication
forks, we performed immunoprecipitation
experiments combined with mass spectrometry and
established that DNA polymerase kappa (Poli) can
partner with MSH2, an important mismatch repair
protein associated with hereditary non-polyposis
colorectal cancer. We found that depletion of
MSH2 impairs PCNA monoubiquitination and the
formation of foci containing Poli and other TLS
polymerases after UV irradiation of cells.
Interestingly, expression of MSH2 in
Rad18-deficient cells increased UV-induced Poli and REV1
focus formation without detectable changes in
PCNA monoubiquitination, indicating that MSH2
can regulate post-UV focus formation by specialized
DNA polymerases in both PCNA
monoubiquitination-dependent and -independent fashions.
Moreover, we observed that MSH2 can facilitate
TLS across cyclobutane pyrimidine dimers
photoproducts in living cells, presenting a novel
role of MSH2 in post-UV cellular responses.
INTRODUCTION
Translesion DNA synthesis (TLS) is a mode of DNA
damage tolerance that uses specialized DNA polymerases
to support DNA synthesis past a spectrum of template
strand base damage, thereby preventing stalled replication
forks from collapse and possible cell death (1). Ten
different specialized DNA polymerases in mammalian cells
have been shown to support TLS in vitro. These
enzymes are devoid of 30!50 proofreading exonuclease
activity and replicate undamaged DNA in vitro with low
fidelity and weak processivity (2). Among them, DNA
polymerases kappa (Polk), iota (Poli), eta (PolZ) and
REV1 belong to a novel DNA polymerase family (the
Y-family) (3,4).
Each of the Y-family polymerases exhibits a preference
for the replicative bypass of specific types of base damage
in DNA. For example, Polk and PolZ support accurate
bypass of benzo[a]pyrene diol epoxide guanine adducts
(B[a]P-dG) and solar ultraviolet (UV) radiation-induced
cissyn thyminethymine cyclobutanepyrimidine dimers
(CPDs), respectively (2,5,6). PolZ-deficient cells manifest
UV radiation-induced mutagenesis, and a markedly
elevated predisposition to UV radiation-induced skin
cancer has been observed in PolZ-deficient humans and
mice (3,4,7). Similarly, Polk-deficient mouse embryonic
fibroblasts and embryonic stem cells are sensitive to the
killing effects of benzo[a]pyrene, and exhibit enhanced
benzo[a]pyrene diol epoxide-induced mutagenesis (810).
Additionally, increased spontaneous mutagenesis has been
observed in some tissues from Polk-deficient mice (9),
suggesting that some, if not all TLS polymerases, are
required for the maintenance of genome stability.
These observations notwithstanding, over-expression of
some specialized DNA polymerases results in elevated
genomic instability that manifests with enhanced
mutation rates as well as DNA strand breaks (3,11,12).
Given the low fidelity of TLS polymerases copying
undamaged templates in vitro, it is believed that TLS
processes in vivo are strictly regulated to keep TLS
polymerases mainly functioning at their cognate substrates in
an error-free fashion. Consistent with these observations,
dysregulation of Polk recruitment to replication forks
promotes genomic instability (13).
TLS in mammalian cells is promoted by
monoubiquitination of proliferating cell nuclear antigen
(PCNA). A number of studies have shown that
monoubiquitinated PCNA exhibits enhanced interaction
with PolZ, Polk, Poli and REV1, relative to unmodified
PCNA (1419). In response to UV radiation, PCNA is
monoubiquitinated at Lys164 by the
ubiquitinconjugating enzyme Rad6 and its cognate ubiquitin
ligase Rad18 (20,21). The upstream signal that activates
PCNA monoubiquitination (PCNA-mUb) in vivo is
replication protein A (RPA)-coated single-stranded DNA
(ssDNA) at sites of stalled forks, in which RPA targets
Rad18 to its sites of action (22). Monoubiquitinated
PCNA is deubiquitinated primarily by the
u (...truncated)