Biology Reference
In-Depth Information
3
0
-OH groups and a non-ligatable 5
0
-deoxyribose pho-
phate (5
0
-dRP) DNA sugar backbone residue. DNA poly-
merase
b
then recognizes and removes 5
0
-dRP and
synthesizes missing nucleotides using the complemen-
tary DNA template. Finally, DNA ligase III seals the
single-nucleotide repair patch in conjunction with the
X-ray repair complementing defective repair in Chinese
hamster cells 1 (XRCC1) protein.
117
MPG knockout
embryonic stem cells are hypersensitive to alkylating
agents.
118,119
Consistently, small interfering RNA
(siRNA)-mediated down-regulation of its expression
hypersensitized HeLa cells to alkylating agents.
120
Inter-
estingly, overexpression of MPG did not show resistance,
but actually sensitized cells to alkylating agents.
121
These
data strongly suggest that proper balance of MPG expres-
sion is critical for its function in the BER of alkylation
damage.
122
In addition to BER, oxidative DNA demethy-
lase, such as hABH2 and hABH3, are also reported to be
involved in N
1
-methyladenine (N
1
-meA), N
3
-methylgua-
nine (N
3
-meG), N
3
-methylthymine (N
3
-meT), and N
1
-
methylguanine (N
1
-meG) DNA repair.
123,124
These two
enzymes belong to the alpha ketoglutarate- and Fe
2
þ
-
dependent dioxygenase super-family, that remove alkyl
groups through an alpha-ketoglutarate-dependent mech-
anism. Although it appears that hABH2 is more efficient
at repairing double-stranded DNAs, and hABH3 prefer-
entially repairs single-stranded RNA substrates, both
hABH2 and hABH3 can repair alkylation DNA lesions
in ssDNA.
124,125
In addition, comparing the responses
of knockout mice revealed that hABH2 was the primary
oxidative demethylase for repairing N
1
-meA and N
3
-
methylcytosine (N
3
-meC) lesions. Furthermore, the
hABH2 knockout increased sensitivity to MMS-induced
cytotoxicity.
126
However, unlike DNA adducts formed
at the N-atoms, other DNA adducts that formed at the
O-atoms such as O
6
-meG in DNA bases are good
substrates for MMR.
104
that can be paired with cytosine (C) or thymidine
(T) after two rounds of DNA replication. hMutS
recognizes these O
6
-meG/C or O
6
-meG/T mispaired
lesions by delineating new versus old DNA. Then,
other components of MMR, such as hMutL, are
recruited and initiate excision of the newly synthe-
sized DNA strand. The MMR-dependent activation
of G
2
checkpoint or programmed cell death is then
activated due to downstream creation of DSBs result-
ing from futile MMR attempts.
128
Since DSBs are
well-known initiators of G
2
arrest and p53-induced
apoptosis in mammalian cells,
129
such a mechanism
seemed plausible in mammalian cells.
106
However,
there appear to be multiple problems in adapting an
MMR-dependent “futile cycling” mechanism to
mammalian systems, among them the apparent lack
of any requirement for p53 signaling for both the G
2
arrest or apoptosis (cell death) responses noted in
MMR-proficient versus MMR-deficient cells.
93,128
In the “direct signaling” model, MMR acts as a DNA
damage sensor and acts to directly signal G
2
cell cycle
arrest and cell death responses by divergent signaling
pathways. The “direct signaling” model was originally
proposed
106,130,131
to explain the MMR-dependent acti-
vation of G
2
arrest and apoptosis, an apoptotic pathway
that later appeared to include c-Abl
101
, as well as the
rapid induction of apoptosis following over-expression
of MMR genes.
103
More recent evidence strongly
suggests that a subset of the MMR proteins (i.e.,
hMSH2, hMSH6, hMLH1, and hPMS2) can serve as
sensors of DNA damage.
128
For example, Hsieh and
colleagues
132
have demonstrated that hMSH2-hMSH6
and hMLH1-hPMS2 specifically bound to a methyla-
tion-damaged O
6
-meG/T mismatched DNA interacted
with Ataxia telangiectasia-and-rad3-related (ATR)-ATRIP
(a PIKK family member) and Chk1. These studies sug-
gested that a direct interaction between MMR and
ATR/Chk1 pathway, which is a part of the controlling
signal transduction pathway for “immediate early” G
2
arrest. Other data (see below) strongly suggest that
c-Abl signaling is responsible for the prolonged G
2
arrest responses, which in turn, activate p73
a
for
lethality (apoptosis).
MMR-Mediated G
2
Cell Cycle Checkpoint
Arrest Responses
Two competing models (futile cycling versus direct
signaling) have been proposed to explain MMR-
dependent cellular responses (i.e., G
2
arrest and cell
death) to specific DNA damage. The data underlying
these two pathways were recently reviewed by us.
6
In the futile cycle model, MMR plays an indirect role
by initiating futile cycles of DNA repair as damage
on the template strand is repeatedly processed, even-
tually leading to the generation of DNA double-strand
breaks (DSBs).
127
Under this model the MMR pathway
plays a single DNA repair function and the repeated
repair events lead to DSBs that result in G
2
arrest
and apoptosis (cell death). Exposure to an alkylating
agent, such as MNNG, creates O
6
-meG DNA lesions
ATR/Chk1 and MMR-Dependent G
2
Arrest
Involvement of the ATR pathway initiating MMR-
dependent signaling pathways for promotion of G
2
arrest remains controversial, and most of our
mechanistic data do not support its role in the MMR-
dependent G
2
arrest and apoptotic responses. The
difference between the data generated to differentiate
roles of ATR versus c-Abl appears to be directly related
to the particular dose of alkylating agents used. Most
available data suggest that under low doses of alkylat-
ing agents, such as MNNG, MMR signals through
