Biology Reference
In-Depth Information
as strong similarities exist between prokaryotic and
mammalian systems.
116
Indeed, most human MMR
proteins have been identified based on their homology
to
E. coli
MMR proteins. These proteins include human
homologs of MutS, MutL, EXO1, single-strand DNA-
binding protein (RPA), proliferating cellular nuclear
antigen (PCNA), pol
d
, and DNA ligase I. MMR in
humans proceeds through the following order of
events.
117
The human homolog of
E. coli
MutS,
hMSH2, forms a heterodimer with hMSH6 or hMSH3
to form hMutS
a
or hMutS
b
, respectively. Both
complexes use the binding and hydrolysis of ATP to
recognize mismatches and initiate their repair. hMutS
a
preferentially recognizes base
However, these apurinic/apyrimidinic sites are typi-
cally recognized and cleaved by AP endonuclease that
creates a SSB at the site of the original lesion. The
resulting SSB can be processed by either short-patch
or long-patch BER.
123
During short-patch repair, a single
nucleotide is replaced whereas 2
10 nucleotides are
replaced during long-patch BER. The choice between
short- and long-patch repair depends upon type of
lesion and the stage of the cell cycle. During short-patch
BER, the primary human polymerase that resynthesizes
DNA is pol
b
, although pol
l
can also perform this
activity. Regardless, DNA ligase III and its cofactor,
XRCC1, catalyzes the nick sealing step during short-
patch BER. DNA synthesis during long-patch BER is
catalyzed by both pol
d
and pol
3
. When these polymer-
ases are coupled with the processivity factor, PCNA,
they are capable of performing strand displacement
synthesis in which the downstream 5'-DNA forms
a flap that is subsequently removed by FEN1. DNA
ligase 1 catalyzes the final ligation step during long
patch BER. It should be noted that defects in BER can
increase the mutation rate in humans which correlate
with the development of cancers
124
and drug resistance
to certain chemotherapeutic agents.
125
Indeed, somatic
mutations in Pol
b
have been found in 30% of human
cancers.
126
e
base mismatches and
mispairs of one or two nucleotides whereas hMutS
b
preferentially recognizes larger mispairs. There are at
least four human MutL homologs designated hMLH1,
hMLH3, hPMS1, and hPMS2 that have been identi-
fied.
118
After recognition of the 3
0
nick and the mismatch,
MutL
a
endonuclease makes an incision 5
0
to the
mismatch in a process that requires PCNA and RFC.
EXO1 is a 5
0
/
e
3
0
exonuclease that performs 5
0
directed
mismatch excision in the presence of MutS
a
or MutS
b
and RPA. After MutL
a
endonuclease makes the 5' inci-
sion, EXO1 performs 5
0
/
3
0
excision from the MutL
a
-
incision site through and beyond the site of the
mismatch. The single-stranded gap that is created by
the exonuclease is then repaired by the activity of pol
d
which uses the other strand as a template. The final
step is sealing of the nick which is catalyzed in an
ATP-dependent process by DNA ligase.
Nucleotide Excision Repair
NER is a versatile multi-step DNA repair pathway
that serves to remove a broad range of bulky, helix-dis-
torting lesions including those formed after exposure
to UV irradiation and anticancer drugs such as cisplatin.
The NER process in mammals is carried out by a multi-
protein complex, commonly referred to as the nucleotide
excision repairosome, that consists of over 30 proteins.
The major steps of mammalian NER include: (a) DNA
damage recognition; (b) assembly of the protein
complex that excises the damaged DNA; and (c) gap-
filling DNA synthesis and ligation of the repaired
DNA.
127
In mammalian cells, NER consists of two
distinct sub-pathways termed global genome repair
(GGR) and transcription coupled repair (TCR). These
sub-pathways are essentially identical except for the
mechanism by which DNA damage is recognized. In
GGR, DNA lesions are recognized throughout the entire
genome by a specific recognition factor denoted as the
XPC
Base Excision Repair
Base excision repair (BER) is primarily responsible for
removing small, non-helix-distorting base lesions from
the genome.
119
A partial list of these DNA lesions
include oxidized bases (8-oxo-guanine), alkylated bases
(3-methyladenine and 7-methylguanine), deaminated
bases (hypoxanthine and xanthine from the deamination
of adenine and guanine, respectively), and uracil that is
inappropriately incorporated in DNA as dUTP or
formed by the deamination of cytosine. Under normal
conditions, this pathway repairs at least 20,000 endoge-
nous DNA lesions per cell per day
120
and this large
number reflects the diversity of DNA lesions that are
processed. The number of DNA lesions can be increased
by orders of magnitude during chemotherapy.
BER is initiated by a wide variety of DNA glycosy-
lases that recognize and excise specific lesions to form
a common intermediate designated as an apurinic/
apyrimidinic site.
121
Since coding information is
removed during this process, these apurinic/apyrimi-
dinic sites are technically non-instructional DNA lesions
that can act as strong blocks to DNA replication.
122
hHR23 complex. After this step, the transcription
factor IIH (TFIIH), XPA and replication protein A (RPA)
sequentially bind to the site of the damage to form a pre-
incision complex. Two helicases, XPB and XPD, which
compose parts of TFIIH, unwind DNA at the site of
the lesion. This allows dual incision of the DNA to occur
which is catalyzed by endonuclease XPG and the
XPF
ERCC1 complex that hydrolyse phosphodiester
e
