Biomedical Engineering Reference
In-Depth Information
with difficulty [
66
-
71
] (discussed later). Although the data are contradictory, it is
possible that MDC regulates checkpoint activation [
98
,
100
,
105
] and binding of
ATM, 53BP1, BRCA1, and other proteins to DSBs, by way of recruiting RNF8
to the sites of lesions and by ubiquitination of H2AX [
106
,
107
, summarized in
98
]. 53BP1 and BRCA1 then push DDR to either NHEJ or HR [
101
,
108
-
110
],
which suggests interconnections not only between NHEJ and ATM signaling, but
also between NHEJ and HR, and shows how individual repair pathways cooperate
and, at the same time, “compete” for DSB substrates. Therefore, the main function
of these mediators that exhibit pleiotropic functions with many partners, is probably
to serve as a scaffold for protein-protein interactions at the sites of DSBs [
101
].
The spectrum of “non-core” proteins recognized to operate in DSB repair is broad
and continues to grow. Many of these proteins participate in reorganization of
chromatin structure at the sites of DSBs to allow their repair, which is discussed
in Section
20.4.3
.
20.3.3
Homologous recombination (HR)
5
0
! 3
0
resection of DNA
ends by MRN complex (MRX in yeasts) and other exonucleases [
60
-
62
,
112
]. This
processing generates long
HR [reviewed,
e.g
., in
85
,
111
,
112
] is enabled by extensive
3
0
-single-stranded DNA (ssDNA) overhangs that mediate
recombination, which are immediately bound by RPA proteins with a high affinity
for ssDNA. RPAs protect DNA from degradation and formation of secondary DNA
structures, as well as from interaction with other HR proteins until they are replaced
with Rad51 in a process mediated by Rad52, Rad55 and Rad57 proteins. Rad51,
the key HR protein, forms a nucleoprotein filament with ssDNA and initiates the
recombination step of HR by invading an undamaged homologous DNA duplex
(stimulated by Rad54, Srs2 helicase, etc.). Dissociation of Rad51 allows strand
pairing and extension of the invading strand by DNA polymerase. The second
3
0
-
DNA overhang at the opposite side of DSB can be captured by the displaced DNA
strand of the donor chromatid (to form a double-Holiday junction) or the extended
invading strand can be released from the complex with the donor chromosome and
anneal with the complementary overhang on the opposite DSB end of the original
chromatid. Holiday junctions are resolved (by resolvase) to yield crossovers or
non-crossovers, and remaining DNA gaps are filled by DNA polymerase while
DNA ligation is accomplished by DNA ligase [
85
]. In synthesis-dependent strand
annealing (SDSA), DSB repair is accomplished as described above; however, only
non-crossovers are produced.
Importantly, while repair fidelity tends to be simplified as “error-prone” NHEJ
and “error-free” HR, HR may in fact yield loss of heterozygosity or mutated
repair products such as translocations and other chromosomal aberrations [
25
,
113
],
because of crossovers between imperfectly homologous (mutated or allelic) loci or
repeated sequences. Thus, repair fidelity is modulated by a precise NHEJ that rejoins
“clear” DSB ends as well as the less precise HR [see
25
for the review,
113
].
