Biology Reference
In-Depth Information
FIGURE 7.2
Principal steps of homologous recombination repair. The process of HRR can be divided into four phases: (1) DNA end
resection; (2) RAD51 filament formation; (3) strand invasion and HJ formation; (4) HJ dissolution. HRR can be completed at the phase 3 via
SDSA, or at the phase 4 via the BLM-TOPOIII complex or HJ resolvases. Depending on how the HJs are resolved, HRR generates either crossover
or non-crossover products.
and BRCA2.
20
e
22
Although BRCA2 can directly bind
ssDNA, both BRCA1 and PALB2 are required for the
formation of BRCA2 and RAD51 foci, suggesting that
BRCA1 is an important regulator of the localization
and function of BRCA2.
that recruits the ATR-ATRIP checkpoint kinase (see
below), and it removes secondary structures of ssDNA.
RPA also inhibits the binding of RAD51 to ssDNA,
which needs to be overcome to enable the subsequent
steps of HRR.
2,3
BRCA2 is the critical mediator for the formation of
RAD51 filament.
16
Human BRCA2 has the ability to
bind multiple RAD51 molecules simultaneously.
17-19
Furthermore, BRCA2 directly binds to single-stranded
overhangs of DSBs. During HRR, BRCA2 plays at least
four biochemically distinct roles to promote the forma-
tion of RAD51 filament: (1) it prevents RAD51 from
binding to dsDNA; (2) it enhances the binding of
RAD51 to ssDNA; (3) it allows RAD51 to overcome the
inhibition of RPA during the binding to ssDNA; (4) it
inhibits the ATP hydrolysis of RAD51, which would
otherwise promote dissociation of RAD51 from ssDNA.
Together, these functions of BRCA2 enable specific and
efficient assembly of RAD51 filament on ssDNA
(
Figure 7.2
).
In human cells, both BRCA2 and RAD51 are localized
to discrete nuclear foci after ionizing radiation (IR), sug-
gesting that they accumulate at sites of DSBs. BRCA2
interacts with a number of proteins including BRCA1
and PALB2, both of which are also required for efficient
HR. PALB2 mediates the interaction between BRCA1
Strand Invasion and Holliday Junction Formation
The formation of RAD51 filament enables ssDNA
overhangs of DSBs to search for homologous sequences
and form the three-stranded intermediate termed the
“synaptic complex”. The synaptic complex allows the
invading DNA strand to anneal with the comple-
mentary strand of the homologous sequences, displac-
ing the other strand to form a “D loop” structure
(
Figure 7.2
).
2
The molecular details of this process are
still largely unclear in human cells. Studies on the bacte-
rial RecA recombinase suggested that the recombinase-
ssDNA filament transiently and non-specifically
interacts with dsDNA, and the interaction between the
two is stabilized when homology is found.
2
Once strand
annealing is accomplished, the 3
0
end of the invading
strand is extended by DNA polymerases (
Figure 7.2
).
The extended DNA strand could dissociate from the
template strand and anneal with the ssDNA overhangs
of the opposite end of DSBs. This process, termed
synthesis-dependent
strand
annealing
(SDSA),
