Biology Reference
In-Depth Information
The Mechanism of NHEJ
In this section we will describe what is currently
known about the mechanism of NHEJ and its role in
the detection and repair of IR-induced DNA damage
as well as in V(D)J recombination.
In its simplest sense, NHEJ can be thought of as
occurring in three stages: (1) detection of the DSB and
tethering of the ends together; (2) processing and
removal of non-ligatable end groups and other forms
of damage surrounding the termini; and (3) religation
of the ends to repair the break (reviewed in
65,66
). The
first step, detection of the break, is carried out by the
Ku heterodimer and the final step, ligation, is catalyzed
by DNA ligase IV within the XRCC4-DNA-ligase IV
complex, however there is considerable uncertainty
about the order of the intervening steps and whether
NHEJ proceeds in a sequential or non-sequential
manner.
109,124,125
Our current understanding of NHEJ
and the roles of the core and accessory proteins will be
discussed below.
subunits exist as a dimer rather than as individual
subunits in the cell. In addition, Ku70 has a unique,
highly acidic N-terminal region of unknown function
and a C-terminal SAP (SAFA/B, Acinus and PIAS)
domain proposed to function in protein
e
protein or
protein
e
DNA interactions (reviewed in
65,129
). Ku80
also contains a unique C-terminal region (Ku80-CTR)
that contains a globular region suggested to be
involved in protein-protein interaction
130,131
and, at
the extreme C-terminus, a region of about 14 amino
acids shown to interact with DNA-PKcs
132,133
(see
Figure 8.6
). The Ku80-CTR protrudes from the DNA
binding core, forming a flexible arm suitable to interact
with DNA-PKcs or Ku molecules bound on the oppo-
site DSB.
134
However, the Ku80-CTR is not absolutely
required for recruitment of DNA-PKcs to sites of UV
laser induced DNA damage
in vivo
suggesting that it
may have additional functions, perhaps recruiting
additional end joining factors to the break.
135
Thus,
Ku may be regarded as a keystone of NHEJ, directly
detecting and tethering DNA ends and being required
for recruitment of DNA-PKcs, XRCC4/DNA ligase IV,
and XLF to UV laser induced DSBs
in vivo
. A recent
study reveals that Ku also has AP lyase activity,
136
sug-
gesting that Ku may also play an enzymatic role in pro-
cessing damaged DNA ends.
Structure and Function of the Core NHEJ Proteins
KU
Ku is an abundant nuclear protein that binds ends of
dsDNA with high affinity and little if any sequence
specificity (reviewed in
126
). Ku is also recruited rapidly
to sites of DNA damage
in vivo
and is required for the
recruitment of DNA-PKcs, XRCC4, and XLF to UV laser
induced DSBs
in vivo
(reviewed in
65,124,127
). Ku is
composed of approximately 70 and 80 kDa subunits
that each contributes to a central DNA binding core
composed of a basket-shaped structure with a pre-
formed ring that can encircle a molecule of dsDNA
128
(
Figure 8. 6
). The extensive interactions between Ku70
and Ku80 (also called Ku86) suggest that the two
DNA-PK
CS
DNA-PKcs is composed of a large, predominantly
a
-helical N-terminal domain followed by a region termed
the FAT (FRAP (FKBP12-rapamycin-associated protein),
ATM, TRRAP (transactivation/transformation-domain-
associated protein)) domain that is weakly conserved
between the PIKK family members and a C-terminal
FAT domain (FAT-C domain) that flanks the kinase
FIGURE 8.6
Domains and structure of Ku70/Ku80.
(A) Domains of Ku70 and Ku80: VWA, von Willebrand
domain; core, core DNA binding domain present in X-ray
structure determined by Walker et al., 2001;
128
SAP, SAF-
A/B, Acinus andPIASdomain; CTR, Ku80C-terminal domain;
DIR, DNA-PKcs interaction region from references.
132,133
(B) The structure of the Ku70/Ku80 core DNA binding
domain with dsDNA.
From Walker
et al.
, 2001.
128
(A)
(B)
