Biology Reference
In-Depth Information
repair activity was observed in
rsc30
and
htl1
deletion strains.
40,73,78
Contrary to
these studies, however, are data from Chai et al. where plasmid repair activity
appeared to be substantially greater than WT in
rsc1
and
rsc2
deletion strains
and in strains with mutant alleles of
sth1
.
72
This contradiction could be
explained by the fact that the efficiency of transformation of uncut and linear-
ized plasmid varies with cell density and that end-joining activity varies with cell
cycle phase and cell density.
79
Overall the data, together with the decreased Ku
recruitment to a DSB in the absence of Sth1, indicate that the RSC complex is
important for enabling efficient end-joining of DSBs.
8. E
FFECT ON
R
EPAIR
:HR
It has been reported that
rsc1
,
rsc2
, and
htl1
mutants are defective in HR/
SSA (single-stranded annealing) in plasmid gap repair assays.
72,78
During
mating-type switching in budding yeast, a DSB introduced at the
MAT
locus
is repaired by HR using one of the silent donor cassettes as a template.
Although strand invasion and extension of the invading strand were unaffected
in an
rsc2
strain, there appeared to be a defect in a postsynaptic step of repair.
Using several assays, we find that recombination is affected in
rsc2
deletion
strains
41
and, given the links between cohesion and recombination and the
defect in cohesion in
rsc
mutants, discussed later, it is tempting to speculate
that the function of RSC in recombination is connected to its effect on
cohesion.
9. C
OHESION
In G1, the cohesin complex (Mcd1/Scc1, Scc3, Smc1, and Smc3) is loaded
onto chromosomes by Scc2-Scc4. During replication, cohesion of the two
sister chromatids is established in a mechanism involving the Eco1 acetyltrans-
ferase, and facilitated by the alternative replication factor C complex containing
Ctf18, Ctf8, and Dcc1, which is important for proper chromosome segregation.
Cohesion remains until chromosome segregation, when it is removed by pro-
teolytic digestion and phosphorylation of Mcd1. In budding yeast, cohesin is
enriched at centromeres and at sites between convergently transcribed genes
on chromosome arms (reviewed in Ref.
80
). Additional cohesin is also recruited
to DSBs, and a large
100 kb domain of cohesin forms around a break in an
H2A S129 phosphorylation-dependent process.
81
Genetic synthetic interactions were found between
rsc2
and several genes
involved in sister chromatid cohesion, namely
scc1-73
,
smc3-4
,
ctf8
,
ctf13
,and
scc2-4
(
eco1)
.
64
To investigate whether RSC contributes to sister chromatid
cohesion, separation of sister chromatids was monitored using GFP-Tet bound
to Tet operator repeats on a chromosome arm. Increased separation of sisters in
G2 was seen in
rsc1
and
rsc2
deletion strains and at the restrictive temperature
in a ts-
sth1
strain.
64,65
Premature separation of sister chromatids occurred
