Biology Reference
In-Depth Information
Resection
Mre11
Ku
Tel1
Mec1
RSC
S129 H2A phosphorylation
Remodeling
F
IG
. 1. The initial binding of some Mre11 and Ku to a double-strand break facilitates remodel
structure of chromatin (RSC) recruitment and remodeling of chromatin immediately adjacent to
the break. The change in chromatin structure promotes binding of more Mre11 and Ku and
subsequently more RSC in a positive feedback loop. The result is efficient resection and H2A
S129 phosphorylation.
6. C
HECKPOINT
A
CTIVATION
The defect in ssDNA formation and H2A phosphorylation in
rsc
mutants
might be expected to lead to reduced checkpoint activity. In support of this,
Liang et al. found reduced Rad9 recruitment to the break in G1 and a small
reduction in Rad53 phosphorylation in response toMMS in
rsc2
cells. However,
when cell cycle arrest following DNA damage was examined directly, we found
that both G1 and G2 checkpoints were activated in
rsc1
and
rsc2
deletion
strains.
41
Moreover, strains containing degron alleles or deletions of the essen-
tial subunits of RSC terminally arrest at the G2/M checkpoint, demonstrating
that RSC activity is not essential for cell cycle arrest. Perhaps checkpoint
activation is modestly delayed in
rsc
mutants, but it appears that the remaining
ssDNA is sufficient to ultimately trigger cell cycle arrest.
7. E
FFECT ON
R
EPAIR
: NHEJ
As may be expected, given its role at a DSB, RSC has been shown to be
involved in the NHEJ pathway. A genetic screen for NHEJ proteins isolated
rsc8
and
rsc30
mutants as novel NHEJ factors.
73
Survival through error-prone
NHEJ of a single DSB at two different loci was decreased in
rsc30
,
rsc1
, and
rsc2
deletion strains,
41,73
and in the case of the
rsc30
deletion strain, altered
sequences were found at junctions. Precise NHEJ was also impaired in this
strain. Another means of assessing end-joining activity is the plasmid repair
assay, in which transformation with a linearized plasmid confers survival on
selective media only if it is successfully repaired by NHEJ. Survival is expressed
relative to that when an undigested plasmid is used in order to normalize for
transformation efficiency. Consistent with a defect in NHEJ, decreased plasmid
