Biology Reference
In-Depth Information
same pathway.
36
It was hypothesized that poly(ADP-
ribosylation) might be necessary for the recruitment of
Ku and DNA-PKcs.
The finding that PARP-1 knockout mice were both
viable and fertile
37-39
was somewhat surprising given
the vital nature of this enzyme in DNA repair until it
was discovered that cells derived from these mice
were capable of catalysing the formation of ADP-ribose
polymers from NAD
fundamental importance of the pathway. It has also been
suggested that the degradation of the ADP-ribose poly-
mers to ADP-ribose units by PARG, combined with the
pyrophosphate liberated by the action of DNA pol
b
,
might provide a local source of ATP, which is necessary
for the completion of the repair process by DNA ligase
III, under conditions of ATP shortage.
26
In addition to covalent modification of proteins, auto-
modified PARP-1 may regulate other DNA-damage sig-
nalling through their association with the polymer.
Several important DNA-damage signalling and repair
proteins (e.g., XRCC1, DNA ligase III, p21, XPA,
MSH6, Ku70, NF-kB, DNA-dependent protein kinase
[DNA-PKcs] and telomerase) have been found to
contain a 20 amino acid poly(ADP-ribose)-binding
sequence motif, consisting of two conserved regions:
a cluster rich in basic amino acids and a pattern of basic
amino acids interspersed with basic residues.
27
The
motif is found in domains that are known to be associ-
ated with protein
in response to DNA damage.
40
Thus, a second DNA-damage activated PARP, PARP-2,
was discovered.
41
PARP-2 can similarly homodimerize
at DNA breaks and it can form heterodimers with
PARP-1 to promote DNA repair.
42
PARP-2 null mice
are also viable but knockout of both PARP-1 and
PARP-2 confers embryonic lethality.
43
PARP-1 is the
major PARP accounting for 80
þ
90% of DNA damage
activated polymer formation and PARP-1 null mice cells
derived from them are sensitive to ionizing radiation
and monofunctional DNA alkylating agents.
38,39,44
Although it is less abundant PARP-2 is also important
in the response to DNA damage, as demonstrated by
the fact that PARP-2 null mice are also hypersentisive
to irradiation and DNA alkylating agents.
43
The “PARP signature” in the NAD-binding site of
PARP-1 is highly (92%) conserved across plant and
animal species
17
with the greatest homology seen with
PARP-2. This “PARP signature” was used to identify
a superfamily of 16 “PARP enzymes”.
22
However, it is
now known that some of these have only mono ADP-
ribosyl transferase activity and some have no known
catalytic activity. Nevertheless vault PARP, tankyrase-1
and -2 and probably PARP-3, are bona-fide PARPs but
PARP-1 and PARP-2 are the only DNA damage acti-
vated PARPs.
In addition to DNA breaks, PARP-1 is also activated
by some forms of supercoiled DNA, such as cruci-
forms,
45,46
and can function as a transcriptional (co-)acti-
vator.
47
However, this is not the subject of this chapter
and the reader is directed at other excellent reviews on
the subject.
48
e
protein interactions, DNA binding,
nuclear localization, nuclear export and protein degra-
dation, suggesting a role for PARP-1 in the regulation
of these processes. Indeed, some of these proteins are
involved in the repair of DNA double-strand breaks
(DSBs) and PARP-1 is involved in this process but its
role is less well defined. The earliest evidence for
a role in DNA DSB repair came from the observation
of Benjamin and Gill in 1980 that the most potent acti-
vator of PARP were blunt-ended DNA double-strand
ends in an
in vitro
system.
28
More recently, this was
confirmed by Haince
et al.
,
29
who also showed that
PARP-1 is necessary for accumulation of MRE11 and
NBS1 at the site of DSB. In cell-based studies inhibition
of PARP by NU1025 retarded the rejoining of IR-induced
DNA DSBs.
30
Moreover, DNA-PK, an important compo-
nent of the non-homologous end joining pathway
(NHEJ) of DNA double-strand break repair (see below),
may be stimulated by PARP-1.
31
Studies showing syner-
gistic radiosensitization by the combined use of PARP
and DNA-PK inhibitors and that inactive PARP-1
inhibited DNA-PK activity, and vice versa, suggest
either loss of mutual stimulation or competition of the
two enzymes for the DNA break.
32,33
In cell-free studies
the synopsis of DNA DSB can be achieved by a complex
of PARP-1, XRCC1, and ligase III (with the further
requirement for PNK for joining of 5-OH termini)
34
and this has been implicated in an alternative non-
homologous end joining DSB repair pathway as
a back-up to the classical NHEJ pathway involving KU
and DNA-PKcs. In these studies PARP-1 competed
with Ku for the DNA ends.
35
However, investigation
of the repair of DNA DSB in cells lacking PARP-1 or
DNA-PKcs treated with PARP or DNA-PK inhibitors
or the combination of inhibitors suggested that both
PARP-1 and DNA-PKcs had equivalent
e
RATIONALE FOR AND DEVELOPMENT
OF PARP INHIBITORS
PARP inhibitors were originally developed to probe
the function of PARP-1, but this avenue of research
was clearly linked to cancer in that the first hint of the
existence of PARP came from investigations of the mech-
anisms underlying the newly developed anticancer
alkylating agents (see above). Based on the observation
that nicotinamide (
Figure 4.2
), the byproduct of the
PARP reaction, had weak PARP inhibitory activity, the
benzamide analogues were synthesized,
49
including
3-aminobenzamide (3AB ) which is still used as a tool
today. The first study revealed that 3AB retarded DNA
roles
in
DNA DSB repair and that
they co-operated in the
