Biology Reference
In-Depth Information
sensitivity to a range of DNA-damaging agents including methyl methane-
suphonate (MMS), phleomycin, bleomycin, hydroxyurea (HU), UV, and
IR.
35,41,70-72
D. The Role of RSC in DSBR
1. R
ECRUITMENT TO
DSB
S
Direct involvement of RSC in the repair of DSBs was proposed when it was
found that Sth1 was recruited to a persistent site-specific break. Using the HO
system described in the introduction, ChIP experiments showed enrichment of
Sth1 relative to an uninduced sample on both sides of the DSB as early as
10 min after HO induction and, as such, this places RSC recruitment very early
in the response to DSBs.
72,73
Sth1 at the
MAT
locus had returned to back-
ground levels 2 h after break formation was switched off.
73
Rsc8 and Rsc1
subunits were also recruited to breaks with comparable kinetics, suggesting
that the whole RSC complex is recruited.
73,74
However, a subunit of the SWI/
SNF complex, Snf5, was detected at the break only after 40 min, that is, later
than RSC, revealing distinct temporal profiles of association for different
remodelers.
72
Enrichment of the Rsc2 isoform of the RSC complex has not
been shown directly but Sth1 recruitment is defective in an
rsc2
strain,
although presumably the much less abundant Rsc1-isoform is still recruited
under these conditions.
72
On the other hand, the Rsc30 subunit is not essential
for Sth1 recruitment.
73
Association of Sth1 or Rsc1 with chromatin is not
impaired in strains in which S129 of H2A cannot be phosphorylated, nor is it
affected by cell cycle phase.
74,75
Collectively, these data suggest that RSC
directly influences DSBR by acting at the break site and that it is required
early in repair.
2. F
ACTORS
A
FFECTING
RSC R
ECRUITMENT
Mre11 and Ku are two of the earliest proteins to bind at a DNA DSB. In
the absence of Mre11, Sth1 enrichment at a DSB was not detectable by ChIP
while its enrichment was slightly delayed in the absence of yKu70.
73
It has also
been reported than Mre11 can interact with Rsc1 and Rsc2.
73
However, as
discussed later, RSC remodeling activity was found at a DSB in the absence of
Mre11, indicating that there is at least a small amount of RSC present. Notably
in that regard, the ChIP data were calculated to determine the enrichment
upon break induction, that is, the amount of RSC bound following break
induction relative to that bound in the absence of the break. As such, a lack
of enrichment does not mean the absence of RSC, rather that there was no
further increase in the amount that was there originally. Consistent with
association prior to the induction of a DNA DSB, we found that Rsc2 and
