Biology Reference
In-Depth Information
junction.
132
After this step, more DNA synthesis occurs
on the invading strand to restore the strand on the
homologous chromosome that was displaced during
the strand invasion step.
bonds 2
24 nucleo-
tides upstream of the damaged site, respectively. The
formed single-strand DNA gap is filled in by pol
d
and/or pol
3
in a process that requires PCNA and
replication factor C (RFC). Finally, the DNA fragments
are ligated by DNA ligase I (LIG1). In TCR, RNA poly-
merase II stalls at the DNA lesion and serves as a signal
to recruit these DNA repair proteins.
8 nucleotides downstream and 15
e
e
Non-Homologous End Joining
The simplest form of NHEJ occurs via the direct
ligation of the broken ends of DNA that occur after
a break. NHEJ requires several distinct proteins
including the Ku 70/80 heterodimer, DNA-dependent
protein kinase (DNA-Pkcs), X-ray-cross-complementing
gene 4 (XRCC4), DNA ligase IV, Artemis, and XRCC4-
like factor (XLF).
133
DSB repair is initiated by detection
of the break by the Ku complex. After binding, the Ku
complex translocates away from the break to allow other
proteins to bind the free end of the DNA break. Ku
recruits DNA-PKcs to the DNA, and the two DNA-
PKcs interact to bridge the DNA ends. This process is
aided by the phosphorylation of DNA-PKcs which func-
tions to protect the DNA ends from non-specific and
excessive degradation. Remodeling of the DSBs is cata-
lyzed by the endonuclease activity of Artemis which
forms single strand DNA gaps that are then filled in
by pol
l
or pol
m
. Once the gap is filled in, XRCC4/LigIV
catalyzes ligation of the nicks. In lymphoid tissue,
a specialized DNA polymerase, denoted as terminal
deoxynucleotidyl transferase (TdT), is sometimes
employed to randomly incorporate nucleotides at the
ends of DNA.
134
This unique activity is used to generate
immunological diversity during V(D)J recombination.
135
In contrast to homologous recombination, NHEJ is more
error-prone as genetic information can be lost from the
ends during end-processing. Thus, mutations and/or
genomic instability such as chromosomal translocations
can occur as an adverse consequence of NHEJ.
136
Repair of DNA Strand Breaks
DNA double-strand breaks (DSBs) result from
normal cellular processes including replication fork
stalling upon encountering endogenously formed
DNA lesions.
128
In addition, DSBs form after exposure
to exogenous DNA damaging sources including
ionizing radiation and anticancer agents such as etopo-
side and doxorubin. DSBs can be repaired through
homologous recombination (HR) or by non-homologous
end-joining (NHEJ), and the decision to proceed using
either repair pathway is determined primarily by the
phase of the cell cycle.
129
In general, HR predominates
during and shortly after DNA replication when sister
chromatids are available (S- and G2 phases of the cell
cycle).
130
In contrast, NHEJ occurs primarily prior to
DNA replication during the G1 phase of the cell cycle.
131
Homologous Recombination
After formation of a DSB, the MRN complex binds to
DNA on either side of the break. The DNA around the 5'
ends of the break is then excised in a process composed
of two distinct steps. In the first step, the 5' ends on
either side of the break are trimmed to create short 3'
overhangs of single-strand DNA. In the second step,
the 5'
3' resection is continued by the Sgs1 helicase
to unwind DNA. The nucleases, Exo1 and Dna2,
degrade the single-strand DNA produced in the first
step. RPA, which has high affinity for single-stranded
DNA, binds the 3' overhangs. The Rad51 protein then
forms a filament of nucleic acid and protein on the single
strand of DNA coated with RPA which then begins to
search for DNA sequences similar to that of the 3' over-
hang. After identification of a complementary sequence,
strand invasion occurs as the single-stranded nucleopro-
tein filament invades the similar or identical recipient
duplex DNA. In human cells, the recipient DNA duplex
provides a template for repair that is generally a sister
chromatid whose sequence is identical to that of the
damaged DNA. A displacement loop (D-loop) is formed
during strand invasion between the invading 3' over-
hang strand and the homologous chromosome. After
strand invasion, pol
d
and/or pol
3
extends the end of
the invading 3' strand to synthesize a new piece of
DNA. This leads to the conversion of the D-loop into
a
/
Translesion DNA Synthesis
Despite the existence of these sophisticated DNA
repair pathways, some DNA lesions escape detection
and persist under cellular conditions. Since these lesions
might block DNA replication catalyzed by high-fidelity
DNA polymerase, there are a number of specialized
DNA polymerase that can bypass lesions in a process
known as translesion DNA synthesis (TLS). The pres-
ence of these specialized DNA polymerases in both
prokaryotes and eukaryotes implies that replication
blockage is a general biological problem that must be
dealt with by inserting a correct or incorrect dNTP oppo-
site a lesion. Thus, the primary function of TLS is
proposed to be as a mechanism to rescue cells from
replication arrest that would otherwise cause cell death.
There are two currently accepted models for how the
activities of various DNA polymerases are coordinated
during TLS (
Figure 5.8
).
137,138
In model A, the replicative
cross-shaped structure known as
a Holliday
