Biology Reference
In-Depth Information
The Mre11-Rad50-Xrs2 (MRX) complex in yeast and the homologous
MRE11-RAD50-NBS1 (MRN) complex in higher eukaryotes recognize
DSBs.
61-65
Even though MRE11 possesses an inherent 3
0
!
5
0
nuclease activ-
ity, the initial 5
0
!
3
0
resection is initiated by the Sae2 (CtIP, in mammals)
endonuclease in complex with MRX/MRN. Sae2/CtIP appears to process
possible adducts on DSB ends for other nucleases to act upon.
66-68
The cell
cycle-mediated phosphorylation of Sae2/CtIP by cyclin-dependent kinases
(CDKs) appears to determine the choice between HR and NHEJ.
69,70
In
mammalian cells, CtIP is ubiquitinated by BRCA1 during S and G2 phases of
the cell cycle, which appears to facilitate its association with DSB sites.
71,72
For extensive resection of the 5
0
strand, the Exo1 and Dna2 nucleases as
well as the Sgs1-Top3-Rmi1 (STR) complex (BLM-TOP3
a
-RMI1-RMI2 in
mammals; Ref.
73
) are recruited.
74,75
The MRX complex is implicated in direct
recruitment of Exo1 and Dna2.
75
Deletion of Exo1, Dna2, or Sgs1 leads to
reduction of resection and the generation of poor HR substrates.
75,76
Further-
more, deletion of EXO1 in mammalian cells causes impaired recruitment of
RPA and ATR at the DSB sites.
77
The initial resection complex that includes
the MRX along with STR and Dna2 has been reconstituted
in vitro
.
78,79
Even
though Top3 and Rmi1 stimulate resection by recruiting Sgs1, they are not
required for the 5
0
strand resection processes.
78,79
Stimulation of Sgs1 helicase
activity by RPA occurs in a species-specific manner, as the bacterial SSB is
unable to stimulate Sgs1.
78-80
RPA also suppresses the inherent 3
0
endonucle-
ase function of Dna2 while stimulating the 5
0
!
3
0
exonuclease required for
DSB resection.
78,79
Two functional human resection complexes have been
reconstituted
in vitro
, one comprising MRN-EXO1-BLM-RPA and the
other MRN-DNA2-BLM-RPA.
80
BLM exhibits direct protein-protein inter-
actions with both EXO1 and DNA2.
80,81
Furthermore, the nuclease activity of
EXO1 is stimulated by BLM, RPA, and MRN.
In the absence of a recombinase or a homologous sequence, resection could
continue over several thousand nucleotides at a rate of approximately 4 kb/h in
yeast.
75,82
During meiotic recombination, resection tracts average
850 nt.
83,84
However, the resection length required for recombination between sister
chromatids during mitotic recombination has not been determined.
82
III. RAD52 Epistasis Group
Many of the proteins involved in the RR pathway are genes of the
RAD52
epistasis group (
Table I
). The name was derived from radiation sensitivity
genetic screening analysis of budding yeast.
85-88
Among eukaryotes, this
group of genes is structurally and functionally conserved.
