Biology Reference
In-Depth Information
the experimental findings that led to the discovery and
elucidation of the NHEJ pathway.
part to the immune system specific process of V(D)J
recombination. V(D)J recombination can be regarded
as the site-specific cleavage and rearrangement of
segments of the immunoglobulin and TCR genes. The
process is initiated by the action of the recombinase acti-
vating genes RAG1 and RAG2 which create DSBs at
specific regions in the IgG and TCR genes that are
then rejoined by components of the NHEJ pathway.
Details of the V(D)J recombination pathway will be dis-
cussed in more detail below and readers are also
directed to several excellent in-depth reviews on the
topic.
101,102
One of the first clues to the links between V(D)J recom-
bination and NHEJ arose from the finding that severe
combined immunodeficient (SCID) mice, which lack
T and B cells, contain a mutation in DNA-PKcs that
destabilizes the protein, crippling DNA-PK-dependent
reactions crucial for V(D)J recombination
103
(discussed
in more detail below). This observation also provided
an explanation for an earlier observation that SCID
mice are radiosensitive.
104
Subsequently, mice lacking
Ku were also shown to be radiosensitive and defective
in V(D)J recombination.
105
Together these findings
brought together DSB repair and V(D)J recombination.
Much of what we now know about NHEJ has thus
been inferred from studies on V(D)J recombination,
and the two fields have developed hand-in-hand for
the last decade or more. For example, XRCC4 was first
identified as an X-ray cross complementing gene
involved in DSB repair
106
and later shown to be required
for V(D)J recombination.
107
XRCC4 is regarded as a scaf-
folding protein that interacts with and stabilizes DNA
ligase IV,
108
the activity of which is essential for both
NHEJ and V(D)J recombination (reviewed in
65,109,110
).
In 2002, Artemis was identified as the gene responsible
for a radiosensitive form of SCID (SCID-A or RS-SCID)
in populations of Native Americans.
111
Artemis is a
nuclease that interacts with DNA-PKcs and functions
in both NHEJ and V(D)J recombination (reviewed
in
112,113
). The most recently identified member of the
NHEJ family, XLF (also called Cernunnos), was discov-
ered in 2006 as the gene defective in patients with radio-
sensitivity and immune deficiency.
114
Simultaneously,
XLF was discovered as an XRCC4-interacting factor
required for NHEJ.
115
XLF is structurally similar to
XRCC4
115
e
117
and interacts with XRCC4 to regulate
DNA ligase IV activity.
118
e
122
Thus, all of the core
NHEJ factors (Ku DNA-PKcs, XRCC4, DNA ligase IV,
XLF and Artemis) are characterized by having roles in
both the repair of IR-induced DSBs and in V(D)J recom-
bination. Animals lacking any one of these core NHEJ
factors are both radiosensitive and immune-deficient
and patients with mutations in DNA ligase IV, DNA-
PKcs, Artemis and XLF are characterized by immune
deficiency and radiation sensitivity.
123
A Historical Perspective on NHEJ
Historically, much of what we know about NHEJ
arose from the identification of rodent cell lines that
were sensitive to X-rays and other DNA damaging
agents.
89
e
91
An important breakthrough came in 1994
when Stamato and colleagues showed that the radiation
sensitive rodent cell line
xrs6
lacked a dsDNA end-
binding activity attributed to the Ku heterodimer.
92
Ku had been known for many years as a protein that
bound double-stranded (ds) DNA in an end-dependent
manner with little or no requirement for DNA sequence
specificity. However, no functional role in DSB repair
had been described. Ku was also known as an autoanti-
gen since antibodies to Ku are found in patients with
autoimmune disorders such as lupus and scleroderma
overlap syndrome. Indeed, the name Ku comes from
the initials of a patient in which the autoimmune anti-
bodies were first discovered and these autoantibodies
proved very useful in the initial purification and charac-
terization of the Ku protein.
93,94
Stamato's identification
of Ku70/80 as the missing factor in radiation sensitive
xrs6
cells set the stage for the characterization of the
genetic defect in other radiosensitive cell lines and
elucidation of what is now referred to as the NHEJ
pathway.
Also in the early 1990s, Anderson and Dynan and
colleagues identified Ku as a component of a DNA-
activated protein kinase activity, termed DNA-PK. In
these initial studies the precise nature of DNA-PK
was still unclear but fractions enriched for DNA-PK
activity were associated with a large polypeptide
termed p350 as well as with Ku.
95,96
At the same
time, p350 was independently identified by Carter
and colleagues as a DNA-activated protein kinase.
97
The situation was clarified in 1993 when it was shown
that Ku was the DNA binding subunit of DNA-PK and
served to recruit p350 to DNA ends, resulting in stim-
ulation of DNA-PK kinase activity.
98,99
p350, which is
now referred to as DNA-PKcs, belongs to the PIKK
family of serine/threonine protein kinases that also
includes ATM and ATR.
78
Confirmation of the role of
DNA-PK in DSB repair came from the finding that
the highly radiosensitive human cell line, M059J,
lacked DNA-PKcs protein.
100
The protein kinase
activity of DNA-PK is now known to be essential for
NHEJ (discussed in detail below).
These early developments in understanding the basis
of cellular X-ray sensitivity proceeded hand in hand
with studies to elucidate the molecular mechanisms of
antibody diversity in the vertebrate immune system.
In vertebrates, sequence variation of immunoglobulin
(IgG) and T-cell receptor (TCR) genes is due in large
