Biology Reference
In-Depth Information
Antimetabolites
This class of compounds comprises modified DNA/
RNA bases and nucleosides. Several antimetabolites are
commonly employed as chemotherapeutic agents. One
of the oldest, 5-fluorouracil, is used as a first-line treat-
ment for advanced colorectal cancers,
59
and its oral pro-
drug, capecitabine, is now being used extensively in
breast cancer treatment.
60
Gemcitabine, a fluorinated
deoxycytidine analog, is licensed for the treatment of
pancreatic cancer, and the fluorinated adenosine
analogue, fludarabine, is highly effective in the treat-
ment of chronic lymphocytic leukemia. Antimetabolites
exert their cytoxicity by a variety of means, including
interference of
de novo
synthesis of nucleosides and
nucleotides, and, after metabolic conversion to their
respective nucleoside triphosphates, termination of
DNA strand elongation either by incorporation into
nascent DNA or as inhibitors of DNA polymerases.
Some of these drugs also have the capacity to inhibit
ribonucleotide reductase, thereby depleting deoxyribo-
nucleotide levels. Blocks to DNA replication can induce
DSB formation and as a result DSB repair pathways
have been implicated in the cellular responses to these
drugs. Although both major DSB repair pathways (dis-
cussed below) can play a role in the response to antime-
tabolites, the choice of pathway may depend on the
drug. Thus, NHEJ appears to be responsible for repair
of fludarabine-induced DSBs,
61
while the resistance
to a new antileukemic agent, 2
0
-C-cyano-2
0
-deoxy-1-
b
-
D-arabino-pentofuranosyl-cytosine (CNDAC), and to
6-thioguanine depend on the major alternative DSB
repair pathway, homologous recombination (HR).
62,63
Interestingly, an additional property of gemcitabine is
its capacity to inactivate HR.
64
complementarity (termed microhomology) at the
termini. Repair by NHEJ frequently results in loss of
nucleotides from either side of the DSB junction, and
NHEJ is frequently refered to as an error-prone pathway.
Despite this, it has been estimated that in mammalian
cells NHEJ is responsible for the repair of up to 85% of
the IR-induced DSBs.
67
NHEJ is active in all phases of
the cell cycle
68
and is the major pathway for the repair
of IR-induced DSBs in both G1 and G2.
67,69
The core
proteins required for the NHEJ pathway are the Ku70/
80 heterodimer, DNA-PKcs (previously called p350),
Artemis, XRCC4, DNA ligase IV and XRCC4-like factor
(XLF, also called Cernunnos) (
Figure 8.5
). As discussed
below, each of these proteins is also required for repair
of endogenous DSBs introduced during the develop-
ment of T and B lymphocytes during the process of
V(D)J recombination, and animals lacking functional
components of the core NHEJ pathway are both
radiation-sensitive and immune-deficient due to defects
in general DSB repair and V(D)J recombination (dis-
cussed in detail below).
IR-induced DSBs can also be repaired by homologous
recombination (HR), a pathway that requires extensive
sequence homology at the DNA ends and results in
accurate repair. In HR, an intact sister chromatid is
used as a template from which to repair the damage,
thus HR is active only after DNA replication, when
a sister chromatid is available.
70
e
72
Although this
pathway will not be discussed in detail here, we will
provide a very brief overview of the process of HR
and the reader is referred to several excellent recent
reviews for in depth coverage of this pathway.
70
e
72
HR
is initiated by recruitment of the Mre11, Rad50, Nbs1
(MRN) complex to DSBs.
73
e
75
The nuclease activities
of Mre11 along with other critical nucleases including
CtIP and Exonuclease 1 and other proteins leads
to 5
0
-3
0
resection of the DNA ends to produce long
3
0
-regions of single stranded (ss) DNA which are then
bound by the ssDNA binding protein RPA. In processes
that are not well understood, the breast and ovarian
cancer susceptibility protein BRCA2 displaces RPA
and facilitates the loading of Rad51 onto the long ssDNA
filaments. Rad51 protein-DNA filaments then invade the
sister chromatid searching for regions of sequence
homology leading to accurate, template directed repair
(see
Figure 8.5
).
Recently a third DSB repair pathway, termed alterna-
tive or Alt-NHEJ (also called microhomology mediated
end joining), has been described. Little is known about
alt-NHEJ save that it occurs in cells lacking components
of the classical NHEJ pathway and involves PARP, DNA
ligase III and XRCC1
76,77
(see
Figure 8.5
).
Nuclear DSBs also trigger an extensive DNA damage
signaling response that activates cell cycle checkpoints
and regulates a host of other cellular responses to
DETECTION AND REPAIR OF DNA
DOUBLE-STRAND BREAKS
IN MAMMALIAN CELLS
The Major DSB Repair Pathways in Mammalian
Cells
As discussed above, IR-induced DSBs are complex
lesions with overhanging ends of differing sequence
and length that frequently contain non-ligatable end
groups. In human cells, the major pathway for the repair
of IR-induced DSBs is non-homologous end joining
(NHEJ).
65,66
As discussed above, NHEJ also plays
a role in the detection and repair of DSBs formed by
exposure to other DNA-damaging agents, in particular
certain chemotherapeutic agents. In NHEJ, DSBs are
ligated with minimal end processing and without
requirement for significant regions of DNA sequence
