Biology Reference
In-Depth Information
four
a
helices, and the C-terminal domain has six to
seven
a
helices. The hallmark of these proteins is the
helix
motifs that are the hallmarks of Nei glycosylase family,
DNA glycosylase activity and the
in vivo
repair function
of NEIL3 remain unclear.
57
helix structural element, followed by
a highly conserved glycine/proline-rich loop.
48
Struc-
tural studies of HhH-containing DNA glycosylases in
complex with DNA have shown that the HhH motifs
participate in DNA recognition through interaction
with phosphate and oxygen atoms of the DNA back-
bone.
39.40
MBD4 protein presents an N-terminal
methyl-CpG binding domain and it is specific for T:G
and U:G mismatches within the CpG sequence context.
OGG1 is a bifunctional glycosylase and it is the
primary enzyme responsible for the excision of 8-
oxoG lesions generated from the oxidative damage of
a guanosine as consequence of exposure to ROS,
causing this base to pair with adenine as well as
with cytosine.
50
In addition, this glycosylase can also
catalyze the removal of faPyG oxidative lesion.
51
MYH, the human homolog of
E. coli
MutY, is respon-
sible for the removal of the normal A base mispaired
with 8-oxoG during DNA replication. If an A:8-oxoG
pair is not repaired before the next round of DNA
replication, a C:G to A:T transversion occurs and the
opportunity for repair is lost.
52
NTH1 is a bifunctional
glycosylase, whose preferential substrate is repre-
sented by oxidized pyrimidines and, as with OGG1,
NTH1 excises base lesions only from duplex DNA
where the undamaged strand provides the template
for repair of the damaged strand.
53
hairpin
e
e
Alkyladenine DNA Glycosylase
AAG enzyme is the only glycosylase identified to
date in human cells that excises alkylation-damaged
bases, although other human enzymes exist that
perform different types of alkylation damage repair.
25,58
This glycosylase has a broad substrate specificity and
has been reported to excise at least 12 different damaged
bases, including 3-meA, 7-meG
59
and undamaged
purines.
60
AAG is a structural outlier, with a topology,
unlike any of the other known BER glycosylase
enzymes, consisting of a single
a
/
b
domain in which
an antiparallel
b
sheet is surrounded by
a
helices.
61
Interaction with DNA occurs through a
b
hairpin which
protrudes into the minor grove.
25
AP Endonucleases
Removal of damaged bases fromDNA by DNA glyco-
sylases leads to the formation of AP sites, which can also
arise spontaneously. In addition to this, free radicals and
alkylating agents promote the release of bases by
introducing base modifications that destabilize the
N-glycosylic bond, generating a better leaving group
moiety.
13
AP sites are a threat to cellular viability and
genomic integrity as they can prevent normal DNA repli-
cation and are cytotoxic and mutagenic.
62
In the BER
pathway, cleavage of baseless sites in DNA is performed
by an AP endonuclease. The major human AP endonu-
clease is APE1 protein (also known as Ref-1 or HAP1),
a homolog of bacterial exonuclease III.
63
The functional
relevance of APE1 is underscored by the fact that nullizy-
gous mice for its gene result in early stage embryonic
lethality
64
and that downregulation of APE1 expression
levels in human cells through RNAi leads to AP site
accumulation, reduced cell proliferation and triggering
of apoptosis.
65
e
67
APE1, in a Mg2
Helix-2 Turn-Helix
These enzymes catalyze predominantly the excision
of damaged purine bases using a completely different
molecular scaffold for damage recognition in respect of
HhH glycosylases.
25
The C-terminal domain contains
the H2TH motif, characterized by the presence of
a
/
b
structures, and a zinc finger (Znf) motif which interca-
lates into the minor groove of DNA.
54
NEIL1 and NEIL2
are both bifunctional glycosylases with broad substrate
range and higher affinity for DNA bubble or forked
structures if compared to single- or double-stranded
DNA. For this reason, this class of glycosylases is
supposed to be preferentially involved in repair of
oxidized bases during DNA transcription or replica-
tion.
55
NEIL1 expression is activated during S-phase,
suggesting its use when DNA is being synthesized,
whereas NEIL2 levels are cell cycle independent, impli-
cating its involvement in transcription coupled repair.
NEIL1 has a long C-terminal region not included in
the crystal structure that has been implicated in interac-
tions with Pol
b
and DNA ligase III
a
, suggesting
a regulatory function of this unstructured tail in coordi-
nating the binding of following enzymes in the BER
pathway.
56
Although NEIL3 presents H2TH and ZnF
รพ
-dependent manner,
is able to recognize an AP site and cleave the phospho-
diester bonds 5' to the AP site, leaving a 3'-hydroxyl
group and 5'-deoxyribose phosphate (dRP) termini
flanking the nucleotide gap.
68
Repair at this point can
proceed along one of the two sub-pathways: short- or
long-patch repair (see
Figure 3.2
). The selection of which
pathway seems to be dependent on the state of the AP
site: normal or regular AP sites are repaired through
the short-patch BER pathway, while several sites and
most modified sites are corrected by the long-patch.
69
In short-patch repair, following APE1 incision, DNA
polymerase
b
(Pol
b
) excises the 5'-dRP moiety and
inserts one or two nucleotides.
70
Then DNA ligase
enzyme completes the repair by sealing the nick.
71
The
