Biology Reference
In-Depth Information
breaks and eventually results in apoptosis.
24
Another
common substrate for nonenzymatic methylation are
ring nitrogens of purine residues, where bimolecular
nucleophilic substitution (S
N
2) by alkylating agents
such as methyl methanesulfonate (MMS) and methyl
halides, leads to the formation of large quantities
of 3-methyladenine (3-meA) and 7-methylguanine
(7-meG) lesions (see
Figure 3.1
). 7-meG does not alter
base pairing with C, while 3-meA blocks DNA replica-
tion and is therefore highly cytotoxic.
23
DNA al-
kylation damage is mostly repaired through the
recognition and removal of the lesions by a specific
DNA glycosylase that excises the modified base
creating an AP site, initiating the BER pathway.
25
In
addition, some lesions are repaired through a direct
damage reverse operated by alkyltransferase protein
(AGT or MGMT).
26
cycle that could otherwise cause mutations by mispair-
ing or lead to breaks in DNA during replication. It
is primarily responsible for removing small, non-
helix-distorting base lesions from the genome.
30
The
currently accepted model for the core BER pathway
reveals the presence of 4
5 distinct enzymatic steps
for the repair of damaged DNA.
31
The pathway is initi-
ated by an appropriate DNA glycosylase, which recog-
nizes and specifically removes oxidated, alkylated or
deaminated bases catalyzing the hydrolysis of the N-
glycosidic bond of the damaged nucleoside and then
forming an AP site. This abasic site in the DNA repre-
sents an intermediate product of BER and is a substrate
for an AP endonuclease or a DNA AP lyase, which
hydrolyzes the phosphodiester bond immediately 5' to
the AP site. The resulting single-strand break (SSB) can
be processed by either the “short-patch”, where a single
nucleotide is replaced, or the “long-patch” BER where
several new nucleotides are synthesized by a DNA poly-
merase. Finally, a DNA ligase can complete the repair
process and restore the integrity of the helix by sealing
the single-stranded DNA nick (
Figure 3.2
).
32
In addition
to these enzymes, a number of accessory proteins are in-
volved, such as the X-ray cross-complementation group
1 protein (XRCC1), the poly(ADP-ribose) polymerase 1
(PARP-1), the proliferating cell nuclear antigen
(PCNA), and the heterotrimer termed 9-1-1. These
proteins provide scaffold for core BER enzymes.
e
PR
OTEINS INVOLVED IN B
ER
Endogenous DNA damage occurs too frequently to
be compatible with life and must be efficiently corrected
by DNA repair mechanisms granting a faithful repro-
duction of the genome with a low rate of mutation.
Damage to DNA alters the spatial configuration of the
helix and such alterations can be detected by specific
agents.
13
Once damage is localized, specific DNA repair
molecules bind at or near the site of damage, inducing
other molecules to bind and form a complex that enables
the repair to take.
25
The nature of molecules involved
and the mechanism of repair that is mobilized depend
on the type of damage that has occurred and on the
phase of the cell cycle. Several unrelated processes elim-
inate lesions in DNA but do not involve excision and re-
synthesis of DNA and for this reason these processes are
referred as “direct reversal” (DR). For example, such
a repair process is the major mechanism in repairing
pyrimidine dimers as a result of UV light irradiation
and methylated guanine, cytosine and adenine.
27
All
other DNA repair mechanisms involve the degradation
or removal of at least the damaged nucleotide followed
by a step of DNA re-synthesis. DNA mismatch repair
(MMR) is a system for recognizing and repairing erro-
neous insertion, deletion, and mis-incorporation of
bases that can arise during DNA replication and recom-
bination (for more detail about MMR, see Chapter 9).
28
Nucleotide excision repair (NER) is a particularly
important mechanism by which the cell can prevent
unwanted mutations by removing the vast majority of
UV-induced DNA damage mostly of them in the form
of thymine dimers and 6
DNA Glycosylases
BER is unique among the excision repair processes in
that the individual base lesions are recognized by
distinct DNA glycosylases. These classes of enzymes
flip the damaged base out of the double helix, and cleave
its N-glycosidic bond leaving an AP site.
33
Eleven
different mammalian glycosylases have been character-
ized so far, which can be broadly divided into two
different mechanistic subclasses: the monofunctional
or “pure” glycosylases and the bifunctional glycosylases
(
Table 3.1
). The first group presents only glycosylase
activity, whereas the second also possesses an associated
AP lyase activity that cleaves the phosphodiester back-
bone at the AP site. Monofunctional DNA glycosylases
typically use an activated water as a nucleophile in
attacking the anomeric carbon of the damaged base,
creating a free base and an AP site.
14
The bifunctional
DNA glycosylases have an associated AP lyase activity
(
b
-elimination activity) that incises the strand 3' of the
AP site. They use an activated amine moiety, generally
a lysine side chain or an N-terminal proline, as a nucleo-
phile for substitution of the damaged base, which leads
to the formation of a Schiff base intermediate between
the nucleophilic lysine or proline and the carbon of the
4-photoproducts (for more
detail about NER, see Chapter 6).
29
The BER pathway
is the primary repair system involved in the removal
of endogenous DNA base damage throughout the cell
e
