Biology Reference
In-Depth Information
long-patch pathway involves the displacements of 2
8
bases around the AP site. The resynthesis of the corre-
sponding nucleotides is accomplished by DNA poly-
merase
d
,or
3
, a process dependent upon PCNA, 9-1-1
complex and possibly other undetermined factors.
69
Following nucleotide addition, the Flap endonuclease 1
(FEN1) enzyme acts to remove the dRP-containing dis-
placed strand in patches between 2 and 6 nucleotides.
Complete repair occurs when DNA ligase I restores the
phosphodiester backbone.
72
In addition to AP endonuclease activity, APE1 also
has 3'-diesterase or phosphatase activity, albeit 200-
fold lower than its AP endonuclease activity.
73
APE1
3'-diesterase activity is essential whenever a bifunctional
glycosylase such as OGG1 or NTH1 removes the
damaged base and incises the DNA backbone. Indeed,
the AP lyase actvity of this type of glycosylase differs
from the endonuclease activity of APE1 in that they
cut 3' to the AP site, whereas APE1 cleaves 5' to the
site, leaving behind a 4-hydroxy-2-pentenal residues
attached to the 3' terminus and a phosphate residue at
the 5' end. APE1 with its 3'-diesterase activity excises
the 3'-phosphate creating the 3'-hydroxyl end required
for Pol
b
binding and activity.
74
In addition to its crucial role in the BER pathway,
APE1 also exerts other functions within the cells: it
works as a redox protein stimulating the DNA binding
activity of several transcription factors that are known
to be involved in cancer promotion and progression.
21
Besides, APE1 also acts as a transcriptional repressor
through binding to negative calcium responsive
elements (nCaRE) of PTH and APE1 promoters,
75
and
very recently Tell and colleagues discovered a new
unsuspected function of APE1 in RNA metabolism
which is controlled by the first 33 residues of the
N-terminal domain.
76
error frequency of short-patch base excision repair in
mammalian cell extracts has been calculated to be of the
order of 5
e
10
e
4
, with one nucleotide deletions pre-
dominating.
80
e
81
Mice nullizygous for the
Pol
10
e
gene are
non-viable and die early in embryogenesis.
82
Under
normal circumstances Pol
b
is present in all tissues
although generally only at low expression levels
83
and
without any cell-cycle dependence.
84
Expression levels
of
Pol
b
are most probably kept low in undamaged cells
due to its mis-insertion frequency compared to replica-
tive polymerases, while they can be upregulated in the
event of DNA damage.
85-86
In addition, overexpression
of Pol
b
is mutagenic and confers a radiation-sensitive
phenotype, and many tumor cells overexpress Pol
b
and have decreased BER fidelity.
87
Some human tumors
express dominant negative mutant forms of Pol
b
(I260M
or the polymerase truncated at the C terminus) that inter-
fere with normal BER.
88
As discussed above, Pol
b
removes the blocking 5'-
dRP moiety through a
b
-elimination reaction, after the
insertion of the nucleotide. However, if the dRP formed
after incision of AP endonuclease is refractory to dRP
lyase activity and therefore cannot be processed by Pol
b
, then a polymerase switch may occur with Pol
d
and
Pol
3
continuing the strand synthesis with displacement
of the DNA strand ahead of the polymerase. In this case
the repair is re-routed through the PCNA-dependent
long-patch pathway in which between 2 and 8 nucleo-
tides are removed and replaced.
69
b
DNA Ligases
BER pathway is completed by ligation of the single-
stranded nick in DNA which must be sealed by
a DNA ligase. This enzyme utilizes the energy of phos-
phoanhydride hydrolysis to make a phosphodiester
bond, and for this reason, the majority of DNA ligases
are ATP-dependent.
89
In humans, two DNA ligases
have been implicated in BER: DNA ligase I (Lig I) and
DNA ligase III (Lig III).
90
Lig I plays an essential role
in DNA replication where it is active in joining Okazaki
fragments
91
and its expression is also upregulated
during cell proliferation.
92
In addition to its replicative
role, Lig I has also been implicated in several DNA
repair pathways including both
93
NER and BER
94
path-
ways. Human cell lines containing a partially inactive
Lig I exhibit hypersensitivity to such DNA-damaging
agents such as ionizing radiation and alkylating
agents,
95
e
96
suggesting a role for Lig I enzyme in BER
pathway and in particular in long-patch repair.
In mammals, the
lig3
gene produces two isoforms of
the mature enzyme by alternative splicing.
97
DNA ligase
III
a
(Lig III
a
) is ubiquitously expressed, whereas DNA
ligase III
b
(Lig III
b
) is found only in testes and is
therefore believed to be involved in homologous
DNA Polymerases
Excision of a damaged base and subsequent process-
ing of the resulting AP site by APE1 endonuclease leaves
an intermediate containing a 3'-hydroxyl, suitable for
priming strand resynthesis by a DNA polymerase and
a 5'-dRP that must be removed prior to completion of
repair by a DNA ligase.
77
As mentioned before, the
majority of BER proceeds through the short-patch
pathway in which a single nucleotide is removed and
replaced. In this pathway, both DNA resynthesis and
removal of the blocking 5'-dRP residue are carried out
by Pol
b
, a member of the X family of polymerases.
78
Pol
b
is a single subunit protein of 39 kDa composed by
two domains connected by a hinge region. The C-
terminal domain contains the polymerase activity,
79
while in the N-terminal resides the dRP lyase activity.
70
Pol
b
lacks any intrinsic proof-reading activity and the
