Biology Reference
In-Depth Information
4. H2A S129 P
HOSPHORYLATION
As mentioned in the introduction, one of the earliest events at a DSB is the
phosphorylation of S129 of H2A by Mec1/Tel1. Recruitment of RSC and its
remodeling of the surrounding chromatin occur on a similar timescale to H2A
phosphorylation, raising the possibility that the two events are connected. Rsc1
enrichment to a DSB is unaffected in a mutant in which H2A S129 cannot be
phosphorylated, but H2A S129 phosphorylation is defective in
rsc
mutants,
placing RSC upstream of phosphorylation in the DNA damage response.
74-76
Residual H2A phosphorylation remained in
rsc
mutants, both by ChIP at a
DSB and by Western blot following MMS treatment, but the efficiency of
phosphorylation was diminished. Additionally, enrichment of Mec1 and Tel1
were decreased approximately twofold in
rsc2
strains, consistent with a defect
in H2A phosphorylation.
75
A recent report has also implicated RSC in DSB-
dependent methylation of H3K4 by Set1,
77
suggesting that remodeling of the
chromatin flanking a DNA DSB is necessary for downstream chromatin
modifications.
5. R
ESECTION
Another of the early events in DSBR is the binding of Mre11 and Ku to the
ends of the break. By ChIP, association of Mre11 and Ku70 with the break site
was reduced when Sth1 expression was repressed, suggesting that RSC-
dependent changes in chromatin structure facilitate Mre11 and Ku binding.
74
One of the predicted consequences of defective Mre11 recruitment would be
reduced efficiency of MRX-dependent resection. Resection was slightly com-
promised in
rsc
mutants as measured by quantitative amplification of single-
stranded DNA (QAOS) or cleavage of restriction sites in double-stranded DNA
(dsDNA).
68,76
In agreement with a resection defect, RPA enrichment was
reduced near the break in an
rsc2
strain and a strain in which Sth1 was
repressed, while Rad51 recruitment was slightly delayed.
74,75
Overall, these data suggest a model for the action of RSC at a DSB (see
Fig. 1
). Following DSB formation, a small amount of Mre11 or Ku rapidly
binds to the ends of the break, which facilitates recruitment of RSC either
directly or indirectly, which in turn remodels the chromatin in the region of the
break. The remodeled chromatin is more accessible and permissive for the
accumulation of more Mre11 and Ku, acting in a positive feedback loop to
recruit further RSC. The presence of Mre11 stimulates resection and conse-
quently recruitment of Mec1- to RPA-coated single-stranded DNA and phos-
phorylation of H2A. This amplification cascade means that although resection
and H2A phosphorylation still occur in the absence of RSC, they occur more
efficiently in its presence.
