Biology Reference
In-Depth Information
To date, 16
in vitro
DNA-PKcs autophosphorylation sites
have been identified,
154,156
e
159
but it has been estimated
that there may be over 40 sites in total.
160
Importantly,
many of the DNA-PKcs
in vitro
phosphorylation sites,
including serines 2056, 2612, 2624, and 3205 and threo-
nines 2609, 2620, 2638, 2647, and 3950, are phosphory-
lated
in vivo
in response to DNA damage.
154,157,160,161
In addition, phosphoproteomics studies have revealed
a total of 37
in vivo
phosphorylation sites, suggesting
that DNA-PK is the target of other protein kinases
in vivo
.
162
Mutational analysis reveals that autophosphorylation
of DNA-PKcs plays critical roles in regulating NHEJ and
V(D)J recombination (reviewed in
151,163
and discussed
in detail below). The most well characterized DNA-
PKcs autophosphorylation sites are the ABCDE or Thr
2609 cluster composed of serines 2612 and 2624 and
threonines 2609, 2620, 2638, and 2647 (
Figure 8.7A
).
DNA-PKcs null rodent cells expressing DNA-PKcs
with serine/threonine to alanine mutations at this
cluster of sites are more sensitive to IR than DNA-
PKcs null cells and rejoined DSBs have reduced DNA
end processing in V(D)J recombination assays,
164
sug-
gesting that phosphorylation of this cluster of sites
increases accessibility of DNA ends to nucleases. More-
over, purified DNA-PKcs with alanine mutations at the
ABCDE cluster is defective at autophosphorylation
induced release from Ku
in vitro
,
134,154
suggesting that
the ABCDE/Thr 2609 cluster of sites is important for
regulating the interaction of DNA-PKcs with Ku and/
or DNA. Together, these studies suggest a model in
which DNA-PKcs is recruited to sites of DNA damage
where it undergoes autophosphorylation, resulting in
release from DSBs. In keeping with this model, both
kinase-dead DNA-PKcs and DNA-PKcs with alanine
at the ABCDE/Thr2609 cluster is retained at
in vivo
sites
of DNA damage longer than wild-type DNA-PKcs.
165
Moreover, the ABCDE/Thr2609 autophosphorylation
cluster is located close to the junction of the N-terminal
a
downstream processing enzymes through autophos-
phorylation induced conformational changes.
XRCC4
XRCC4 is composed of a globular head, an elongated
coiled-coil stalk and a CTR of unknown function.
166,167
XRCC4 exists primarily as a dimer, mediated through
head and stalk domain interactions.
166,168
XRCC4 has
no enzymatic function but interacts with, stabilizes
and stimulates the activity of DNA ligase IV.
108,169
XRCC4 also interacts with XLF,
115,167
polynucleotide
kinase/phosphatase (PNKP)
170,171
and DNA
117,119,120,148
and can form extended protein filaments in solution.
167
It has been suggested that these filaments may allow
XRCC4 to interact with DNA, facilitating DNA ligation
by DNA ligase IV.
DNA-LIGASE IV
DNA ligase IV is composed of an N-terminal catalytic
domain linked to a C-terminal tandem BRCT domain.
172
BRCT (BRCA C-terminal) domains are protein
e
protein,
sometimes protein
e
phosphoprotein or protein
e
DNA
interacting domains found in BRCA2 and many other
proteins in the DNA damage response.
173,174
The
tandem BRCT domains in DNA ligase IV interact with
the stalk region of XRCC4 in a high affinity interaction
that stabilizes DNA ligase IV.
175,176
It is likely that all
of the cellular DNA ligase IV exists in complex with
XRCC4.
108
However, XRCC4 is more abundant than
DNA ligase IV in human cells, suggesting that it may
also exist in the absence of ligase IV.
170
XLF
Like XRCC4, XLF is a dimeric protein composed of
a head, stalk, and an unstructured C-terminal domain,
but unlike XRCC4 the stalk domain contains a kink
that reverses its direction.
116,117
The precise function of
XLF is unknown but it has been shown to regulate the
ADP-ribosylation step of the DNA ligase IV reac-
tion1
122,177
and stimulate ligation of non-complementary
DNA ends by DNA ligase IV.
118
-helical domain of DNA-PKcs and the beginning of the
FAT domain, in a region suggested to be located in
flexible hinge regions at the apex of the DNA-PKcs
molecule.
137
Significantly, the solution structure of
DNA-PKcs reveals that autophosphorylation results in
extensive conformational change,
134
thus, we have sug-
gested that autophosphorylation of this region of DNA-
PKcs may regulate opening and closing of the gap at the
base of the DNA-PKcs molecule and thus regulate inter-
action of DNA-PKcs with DNA.
162
Moreover, inability of
DNA-PKcs to autophosphorylate at the ABCDE/
Thr2609 sites delays the initial resection step of HR,
67
suggesting that autophosphorylation regulates initiation
of HR as well as NHEJ. Thus, DNA-PKcs appears to play
a regulatory role in NHEJ, initially joining with Ku to
tether dsDNA ends then providing controlled access to
ARTEMIS
Artemis is composed of an N-terminal nuclease
domain and a C-terminal SQ rich domain of unknown
function that is highly phosphorylated
in vivo.
178,179
Early studies suggested that Artemis had both endonu-
clease and exonuclease activities,
112
but a recent report
suggests that the exonuclease activity is due to a contam-
inating protein.
180
Artemis interacts with DNA-PKcs
suggesting that DNA-PKcs recruits Artemis to sites of
DSBs.
181
The endonuclease activity of Artemis requires
DNA-PKcs protein kinase activity, but this probably
occurs through autophosphorylation of DNA-PKcs
rather
itself.
150
than phosphorylation of Artemis
