Biology Reference
In-Depth Information
6. E
FFECT ON
R
EPAIR
: NHEJ
Given its recruitment and activity at a DSB, it might be expected that
INO80 plays a role in DNA repair. Moreover, mutants in the HR pathway show
synthetic sickness and additive sensitivity with
ino80
mutants.
109,110
Breaks
formed at the
MAT
locus in a strain with the
HML
and
HMR
donor cassettes
deleted can only be repaired by NHEJ. After a pulse of HO expression and
then growth on glucose plates to switch off expression, error-free NHEJ allows
survival. In this assay, a strain with the first 900 bp of
ino80
deleted,
arp8
and
nhp10
deletion strains did not have a defect in error-free NHEJ.
107,110,111
An
arp8
deletion strain also behaved like the WT in a plasmid repair assay, another
assay for error-free NHEJ.
110
Conversely, in the presence of constant HO
expression, by plating on galactose plates (or plates without doxycycline using
a tet-HO expression plasmid), survival is dependent on error-prone NHEJ to
prevent cleavage of the HO site again. In this assay,
arp5
and
arp8
deletion
strains and the
ino80
mutant lacking the first 900 bp of the gene displayed a
mild defect in error-prone NHEJ.
101,107,111
This could indicate a decreased
ability to process DNA ends in
ino80
mutants.
7. E
FFECT ON
R
EPAIR
:HR
As INO80 appears to facilitate resection at a DSB, it may be expected to
affect repair by HR. To initiate HR, single-stranded DNA generated by resec-
tion is first coated with RPA, which is then exchanged for Rad51 that is capable
of homology searching and strand invasion catalyzed by Rad52. Analysis of
recruitment of RPA to the break site in an
arp8
deletion strain with the mating-
type donor cassettes deleted was found to be comparable to the WT, but a slight
delay in Rad51 loading was observed.
110
However, an
arp8
strain with the
donor cassettes present, which is therefore proficient for mating-type switch-
ing, did not have reduced Rad51 at the
MAT
locus, but possessed a decreased
amount of Rad51 at the
HMRa
locus.
115
This
arp8
strain and one with the first
900 bp of
ino80
deleted displayed a delay in strand invasion at
HML
.
111,115
This was attributed to diminished nucleosome repositioning at the
HMRa
locus
in the
arp8
deletion strain. Despite these differences with the WT,
ino80
mutants do not have a dramatic defect in mating-type switching, demonstrating
that efficiency of HR is not affected or compensated.
107,111,115
Mating-type switching is a very specific type of HR, involving gene conver-
sion without a crossover. INO80 has been found to have a role in other types of
recombination. In haploid
arp8
cells, spontaneous sister chromatid recombi-
nation occurred at the same frequency as in the WT, but while the rate of
recombination of the WT strain increased following treatment with MMS, the
rate in the
arp8
strain remained the same.
116
Similarly, the rate of spontaneous
recombination between alleles on different chromosomes is the same for WT
a
