DNA Double-Strand Break Repair by Non-homologous End Joining and Its Clinical Relevance - DNA Repair in Cancer Therapy - page 167

and initiation of repair by HR 73,74 ( Figure 8.5 )andthe
Mre11,Rad50Xrs2(MRX)complexhasawe -
established role in NHEJ in yeast. 192 The role of MRN
in mammalian NHEJ has been controversial but recent
studies suggest that MRN plays a role in both alterna-
tive 193 and classical NHEJ. 193

The function of Artemis' endonuclease activity has been
well established in V(D)J recombination (discussed
below), but its role in repair of IR-induced DSBs is less
clear.

Additional NHEJ Proteins
The core NHEJ proteins described above function in
both repair of IR-induced DSBs and in V(D)J recombina-
tion. In addition, repair of IR-induced DSBs may require
the involvement of DNA end processing factors such as
PNKP and Tdp1 and DNA polymerases that are not
regarded as core NHEJ proteins and are discussed
below.

ATM
As discussed above, the main role of ATM in the
DNA damage response is in the initiation and orchestra-
tion of the extensive DSB induced signaling response
( Figure 8.5 ). Indeed, it has been estimated that at least
85% of DSB repair in human cells occurs independently
of ATM. 194 However, recent studies, primarily from the
Jeggo laboratory, indicate that ATM plays a critical role
in the repair of a small subset (approximately 15%) of
IR-induced DSBs that occur at the periphery of regions
of nuclear heterochromatin. These studies provide an
elegant explanation for the observed biphasic kinetics
of DSB repair in human cells: an initial fast phase of
repair, lasting about 4 hours, which is ATM-independent
and represents NHEJ-mediated repair of DSBs that
occur in regions of low chromatin complexity and
a slower phase of DSB repair, often lasting 24 hours or
more, that represents repair in regions of higher chro-
matin complexity that requires ATM, KAP-1 (KRAB-
domain Associated Protein 1), and Artemis. 67,69,194,195

PNKP
Polynucleotide kinase/phosphatase (PNKP) has both
5 0 -DNA kinase and 3 0 -DNA phosphatase activities and is
thus ideally suited to converting IR-induced 5 0 -OH and
3 0 -phosphate groups to ligatable end groups. 182 PNKP
is composed of an N-terminal FHA (forkhead associated)
domain, a phosphopeptide binding domain found in
many proteins involved in the DNA damage response,
that is tethered to the catalytic domain by a flexible
linker. 183,184 The FHA domain is critical for the interac-
tion of PNKP with CK2 phosphorylated XRCC4 (phos-
phorylated on Thr-233), 171 suggesting that XRCC4
and/or the XRCC4-DNA ligase IV complex recruits
PNKP to sites of DNA damage. PNKP also interacts
with XRCC4 through a phosphorylation independent
interaction with its catalytic domain but the physiolog-
ical significance of this interaction is unclear. 170

PARP3/APLF
A recent study has shown that poly(ADP-ribose)
polymerase 3 (PARP3) recruits aprataxin and PNK like
protein (APLF) to DSBs where it promotes retention of
XRCC4-DNA ligase IV, enhancing DSB repair 196 linking
aprataxin and PARP3 to NHEJ.

TDP1
Tyrosyl-DNA phosphodiesterase 1 (Tdp1), which is
defective in hereditary spinocerebellar ataxia with
axonal neuropathy (SCAN1), catalyses the removal of
stalled topoisomerase 1 from 3 0 -DNA termini and the
conversion of 3 0 -phosphoglycolate to 3 0 -phosphate
groups. 185 As such, Tdp1 is ideally suited to play
a role in removal of non-ligatable IR-induced DNA
lesions. Tdp1 has been shown to function in NHEJ in
yeast, 186,187 but whether it is involved in NHEJ in
mammalian cells is not clear. 188,189

Mechanism of NHEJ: Repair
of IR-induced DSB
The first step in NHEJ is recruitment of the Ku heter-
odimer to ends of DNA ( Figure 8.8 ). Ku has the ability
to translocate along DNA, and this allows for recruit-
ment of DNA-PKcs to the extreme DNA termini to
form the DNA e PK complex. 197,198 Ku and DNA-PKcs
are thought to protect DNA ends from nuclease attack
and also to tether DNA ends together in a synaptic
complex. The DNA-PK complex then undergoes auto-
phosphorylation, probably in trans 161 which results in
a conformational change that either releases DNA-
PKcs from DNA termini or remodels the DNA ends,
providing access to downstream processing enzymes.
Whether DNA-PKcs is released prior to or after recruit-
ment of the XRCC4-DNA ligase IV complex to the DSB
is not yet clear. Experiments using UV laser-induced
damage suggest that DNA-PKcs is not required for

DNA POLYMERASES MU AND LAMBDA
DNA polymerases of the X family, specifically mu
and lambda, likely play DNA gap-filling roles in V(D)J
recombination and DSB repair 190 and the lymphoid-
specific polymerase X family member terminal deoxy-
nucleotidyl transferase (TdT) plays an important role
in V(D)J recombination 191 (see below).

MRN
In mammalian cells the Mre11, Rad50, Nbs1 (MRN)
complex plays critical roles in activation of ATM 81