Biology Reference
In-Depth Information
recombination during meiosis
.
98
The two isoforms share
many features in common and differ only at the C-
termini.
99
Both enzymes contain a putative ZnF motif
which is believed to function as detector of DNA
nicks.
100
DNA Lig III
a
was originally purified in
complex with XRCC1,
101
and further characterization
unveiled the biological relevance of this protein:protein
interaction. XRCC1 null cell
DNA repair and damage response proteins. The protein
is composed by several domains including a DNA
binding domain, an automodification domain and a cata-
lytic domain.
114
Although PARP-1 is ubiquitously found
in complex with BER core proteins, it seems to be
dispensable for BER since the pathway can be reconsti-
tuted from purified enzymes in the absence of
PARP-1.
115
The main role of PARP-1 may be related to
histone poly(ADP)-ribosylation, a modification that facil-
itates the access of BER enzymes to the site of damage,
116
or to recruitment of XRCC1 and Pol
b
through their inter-
action with automodified PARP-1 itself.
117
Proceeding of
BER requires the displacement of PARP-1 from the nick,
which is possible only after its automodification. This
could represent a mechanism of protection for the cells
from chromosome degradation by unrestrained BER if
excessive DNA damage occurs.
118
6-fold
reduced levels and activity of DNA Lig III
a
102
and are
deficient in short-patch BER, suggesting that Lig III
a
is
fundamental in this pathway.
103
lines have a 4
e
Scaffold Proteins Involved in the BER Pathway
All the enzymes discussed thus far constitute the core
BER pathway and are sufficient to reconstitute BER
in
vitro
. Nevertheless, a certain number of accessory
proteins are involved in BER
in vivo
and although they
do not exert a specific enzymatic activity, they provide
a scaffold for the core BER enzymes (see
Figure 3.2
).
PCNA
Proliferating cell nuclear antigen is an accessory
factor required in eukaryotes for efficient replication
by DNA polymerase
d
, and as a consequence, PCNA is
required during the long patch BER as support for
Pol
d
.
119
The active factor is composed by three mole-
cules of PCNA associated to form a homotrimeric
structure which functions as a scaffold protein facili-
tating exchange and recruitment of other factors in repli-
cation forks, in particular in the presence of obstacles
during replicative processes.
120
In addition, PCNA can
also attract BER enzymes in the site of lesions repair
encountered during the replication. Beside Pol
d
,
PCNA is known to interact with other BER enzymes
such as UNG and NTH1 glycosylases,
121
e
122
FEN1,
123
APE1, Pol
b
, and DNA ligase I.
124
e
125
XRCC1
XRCC1 has been increasingly implicated as a key
player in the BER process. Cells deficient in XRCC1
show many of the hallmarks of defective BER including
hypersensitivity to ionizing radiation and alkylating
agents, delayed SSB rejoining and induced mutation.
104
The importance of the
XRCC1
gene for cellular func-
tioning is underlined by the fact that its knockout results
in embryonic lethality.
105
Although this protein has not
known enzymatic function of its own, XRCC1 interacts
with a number of enzymes involved in BER pathway.
As mentioned before, XRCC1 interacts with Lig III
a
101
but not Lig III
b
,
106
with Pol
b
,
107
PARP-1,
108
APE1,
109
OGG1,
110
and PCNA.
111
XRCC1 is a multi-domain
protein with an
N
-terminal DNA binding domain and
two BRCT (BRCA1 C terminal) motifs. The first BRCT
motif is the site of interaction with PARP-1,
108
while the
second one is responsible for the interaction with Lig
III
9-1-1
In addition to PCNA, eukaryotic cells possess another
polymerase clamp, a heterotrimer constituted by Rad9,
Rad1, and Hus1 proteins and named 9-1-1 complex.
126
Its main function probably consists in recruitment of
damage-processing proteins to the sites of stalled repli-
cation. As for PCNA, the 9-1-1 complex has been
demonstrated to interact with and stimulate many BER
proteins, such as MYH,
127
NEIL1,
128
TDG,
129
APE1,
130
Pol
b
,
131
FEN1,
132
and Lig I,
133
suggesting its involve-
ment in replication-associated BER in a mode similar
to that of PCNA.
.
106
The effects of these interactions may either acti-
vate or inhibit the enzymatic activity of the interacting
partner. Functionally relevant are the binding of
XRCC1 with Pol
b
and Lig III
a
. Interaction between
XRCC1 andPol
b
contributes to cellular resistance against
alkylating agent and single-strand break repair.
112
On the
other hand, absence of XRCC1 significantly reduces
the ligation efficiency of Lig III
a
in the short-patch BER
pathway where this DNA ligase is involved.
103
a
PARP-1
Poly(ADP-ribose) polymerase (PARP) are enzymes
that use NAD
รพ
to modify themselves and other proteins
with branched polymeric chains consisting of adenosine
5'-(5'-ribose)diphosphate units.
113
PARP-1 is activated by
binding to nicks in DNA and then modifies a number of
APE1: NOT ONLY A DNA REPAIR
ENZYME
While describing the multistep BER pathway, we
identified APE1 as the major human apurinic/apirimi-
dinic endonuclease. Indeed, human APE1 was first
