Biology Reference
In-Depth Information
CHAPTER
4
The Role of PARP in DNA Repair and its
The
rapeutic Exploita
tion
Nicola J. Curtin, Asima Mukhopadhyay, Yvette Drew, Ruth Plummer
Newcastle University, Newcastle upon Tyne, UK
DISCOVERY OF PARP, STRUCTURE
AND F
UNCTION, PARP SUPERF
AMILY
restricted to a 42 kDa region at the COOH-terminus of
the enzyme. The loss of this 45-amino acid fragment
completely abolishes enzyme activity.
15
Within this
domain are the amino acid residues critical for NAD
þ
binding and polymer formation, the nicotinamide moiety
forms hydrogen bond interactions with Ser904 and
Gly863 and
p
Poly(ADP-ribose) polymerase (PARP) enzymes
catalyse the formation of ADP-ribose polymers using
NAD as a substrate. The product, poly(ADP-ribose),
and the first PARP enzyme were discovered indepen-
dently by scientists in France and Japan in the
1960s.
1
e
6
The observation that it was catalytically acti-
vated by DNA damage explained the earlier observation
that alkylating agents caused a rapid and profound
depletion of NAD
þ
, which had originally been thought
to be due to an effect on glycolysis.
We now know this founder enzyme as PARP-1 (EC.
2.4.2.30); it is the most abundant and best characterized
of a family of PARP enzymes. It is encoded by the
ADPRT-1
gene located on chromosome 1q41
p
-interactions with Tyr904 that are neces-
sary stabilization of the cleaved NAD and Glu988 is crit-
ical for glycosidic bond formation in the growing
chain.
16
This C-terminal catalytic domain contains the
region of highest conservation between species called
the “PARP signature”.
17
PARP-1 is constitutively expressed, with low basal
activity. In response to DNA breaks the zinc fingers of
PARP-1 bind DNA and this activates its catalytic activity
around 100-fold. This involves the breakage of the glyco-
sylic bond between the C-1' atom of ribose and the nico-
tinamide in NAD
þ
, followed by and the formation of
a new glycosylic bond with the nucleophilic acceptor
protein and subsequently the growing ADP-ribose chain
in a progressive fashion to form long linear and
branched homopolymers of ADP-ribose, poly(ADP-
ribose) or PAR. Residues of glutamic acid, aspartic
acid and lysine are acceptors for poly(ADP-ribosyl)ation
on the target proteins (
Figure 4.1
). The main acceptor
proteins are PARP-1 itself and histone H1 but topoiso-
merases HMG proteins and DNA polymerases and
ligases have all been described as targets.
7
e
19
Biochem-
ical studies reveal that PARP-1 acts as a catalytic dimer
20
and DNA footprinting studies demonstrate that PARP-1
protects DNA 7 nucleotides either side of the break
9
sug-
gesting that 2 PARP-1 molecule binds either side of the
break and poly(ADP-ribosyl)ate each other.
PARP-1 plays a fundamental role in DNA single-
strand break repair (SSBR, also sometimes known as
base excision repair, BER). The formation of the highly
negatively charged polymer on histones and PARP-1
e
q42 and
consists of 23 exons spanning 43 kb.
7,8
It has a molecular
weight of 113 kDa and consists of three major domains.
The DNA-binding domain occupies the 42 kDa NH
2
-
terminal region, which includes two zinc-finger motifs
that bind DNA breaks
9
as well as a nuclear localization
signal (NLS).
10
This domain also contains a third zinc
finger which is not necessary for DNA binding but plays
an, as yet undefined role, in activation of the catalytic
activity.
11
Also located in the DNA binding domain is
a BRCA1 carboxy-terminal (BRCT) motif, such motifs
are commonly found in DNA damage response and
cell cycle checkpoint proteins, where they promote
protein
e
protein interactions. The centrally located
16 kDa automodification domain of PARP-1 (between
residues 374
e
525 in the human protein) contains
conserved glutamate and lysine residues, which are the
targets for auto-poly(ADP-ribosyl)ation.
12,13
The 55 kDa
catalytic domain of human PARP is located in the
COOH-terminal region of the enzyme spanning residues
526
e
1014.
14
The ADP-ribose transferase activity is
e
