Biology Reference
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effect of INO80 on histone eviction, as INO80 could indirectly promote histone
eviction by allowing more efficient nuclease activity. This could be achieved
either by histone sliding or by removal of H2AZ as described earlier.
Resection may result in loss of histones, but equally a decrease in histone
ChIP signal would occur if the histones remained associated with single-
stranded DNA. An apparent anomaly exists in that INO80 does not significant-
ly accumulate at a break until 1-2 h after break formation, yet Mre11 recruit-
ment, which is defective in
ino80
mutants, occurs very quickly. This could be
explained by a feedback loop similar to that proposed for the RSC complex,
where some rapid Mre11 and Tel1 binding to ends can trigger H2A phosphor-
ylation, enabling INO80 recruitment and its remodeling activity. Remodeling
could promote resection to allow further recruitment of Mre11 and Mec1.
Therefore, while initial Mre11 binding and H2A phosphorylation would be -
INO80-independent, subsequent retention and accumulation would be
INO80-dependent. As H2A phosphorylation and INO80 have distinct locali-
zation profiles, it may also be the case that initial recruitment of INO80 to the
break site is not via phosphorylated H2A directly, but via another mechanism
and that phosphorylated H2A is then required for INO80 retention. This
hypothetical initial recruitment signal may not be present immediately after
break formation, hence the delay in INO80 recruitment.
5. C
HECKPOINT
A
CTIVATION
Cells containing
ino80
mutations are proficient in checkpoint activation in
response to DNA-damaging agents, but are compromised in response to a
DSB. Cell cycle arrest,
RNR
induction, and Rad53 activation were normal in
response to HU in
ino80
and
arp5
deletion strains (although downregulation of
Rad53 phosphorylation was delayed).
103,109
In response to MMS, Rad53 was
activated, if not hyperactivated, in
ino80
,
arp5
, and
arp8
deletion strains and
MMS-dependent induction of
RNR
genes occurred.
101
In contrast, when
arp8
was deleted, Rad53 activation was impaired following HO cleavage to create a
DSB.
107
This would suggest that resection is required in order to efficiently
activate the checkpoint in response to a DSB, but not in response to treatment
with HU or MMS, which activates the intra-S checkpoint. However, despite the
delay in Rad53 activation in response to a DSB, the checkpoint is eventually
activated sufficiently to arrest the cell cycle at G2/M.
111
INO80 also appears to
play a role in adaptation to checkpoint arrest, which permits continued pro-
gression through the cell cycle, despite the remaining presence of DNA
damage.
111
Finally, Mec1/Tel1 phosphorylation of the Ies4 subunit of INO80
was implicated in modulating a checkpoint in response to replication stress in a
redundant manner to Tof1.
114