Biology Reference
In-Depth Information
that INO80 is not able to catalyze histone eviction
in vitro
. However, a study
from van Attikum et al. did normalize to input DNA and still found loss of H3
after 2-4 h close to the break at the
MAT
locus, and at another locus (
PDR10
),
the H3 signal first increased and then fell to below the level before break
induction.
107
The loss of H3 at the
MAT
locus was slightly delayed in an
nhp10
deletion strain, but this mutant had no effect on the chromatin changes at the
PDR10
locus. It is difficult to reconcile the results from these different studies,
but one possibility is that changes in chromatin structure due to remodeling or
binding of other proteins may mask the epitopes recognized by the antibodies
used in the ChIP assays.
In light of recent biochemical studies of the INO80 complex that found no
detectable nucleosome eviction activity, the role of INO80 at a DSB may
instead be to increase accessibility by either sliding nucleosomes or via histone
exchange.
99
Shortly after DNA DSB formation, SWR1-dependent deposition
of H2AZ was detected at a break.
113
However, this enrichment of H2AZ was
found to be transient and 2 h after break induction, the signal had almost
returned to basal levels.
107,113
Given its ability to replace H2AZ with H2A
and its localization at breaks, INO80 could be responsible for the removal of
H2AZ at DNA DSBs and this conceivably could be important for regulating
chromatin accessibility during repair.
4. R
ESECTION
Single-stranded DNA accumulates at a DSB with similar kinetics to INO80
enrichment and so the effect of INO80 activity on resection was examined.
After DSB induction, less single-stranded DNA was detected in
arp8
and
nhp10
deletion strains than in the WT by a qualitative QAOS assay.
101,107
A decrease in single-stranded DNA was also detected in
arp8
,
nhp10
, and
ino80
deletion strains by Southern blot and restriction enzyme digest assays,
114
while in ChIP assays, slightly more input DNA was amplified from an
arp8
deletion strain than from the WT.
112
Taken as a whole, this suggests that there
is a small but detectable defect in resection in
ino80
mutants. In agreement
with this, 1 h after break formation, Mre11 recruitment to a break was reduced
in an
arp8
deletion. Similarly, Mec1, which binds to RPA-coated single-
stranded DNA displayed reduced association with the break in an
arp8
strain.
107
This defect may not be sufficiently large to be detected in all assays
or strain backgrounds, as no problems with resection were seen in an
arp8
strain or a strain lacking 900 bp from the start of
ino80
in restriction enzyme
assays, although a delay in strand invasion was found in the
ino80
strain.
110,111
Overall,
ino80
mutants appear to have a slight defect in single-stranded
DNA formation, suggesting that INO80 activity at chromatin next to a break
facilitates efficient resection. This could, at least in part, explain the potential
