Biology Reference
In-Depth Information
that can bind acetylated lysines), INO80 types (with a split ATPase domain),
8
ISWI types (characterized by SANT and SLIDE domains), and CHD types
(characterized by chromodomains that can bind methylated lysines). Complex-
specific auxiliary subunits confer different properties to the remodeling com-
plexes, in terms of both regulation of catalytic activity and recruitment through
different chromatin-binding domains. Evidence exists for the involvement of a
number of chromatin-remodeling complexes in the DNA damage response,
such as RSC, INO80, SWR1, and SWI/SNF in budding yeast and PBAF, BAF,
ACF, and TIP60 in mammalian cells (reviewed in Ref.
9
).
The role of chromatin-remodeling complexes in double-strand break repair
(DSBR) has been most extensively studied using the HO system in
S. cerevi-
siae
.
10
Mating-type switching in budding yeast involves introduction of a DSB
at the mating-type locus by HO endonuclease, followed by intrachromosomal
recombination with one of two silent donor cassettes,
HMLa
or
HMR
a. In the
HO system used to study the molecular details of DSBR, the donor cassettes
have been deleted and therefore induction of HO introduces a persistent DSB
at a single, specific location (
MAT
locus) within the genome that cannot be
repaired by HR. Although repair of this break can only occur through NHEJ,
the key proteins involved in HR are still recruited to the break. This allows the
temporal accumulation of repair and checkpoint proteins, histone modifica-
tions, and changes in nucleosome positioning to be followed by chromatin
immunoprecipitation (ChIP) and nuclease accessibility experiments.
One of the earliest events that can be detected following DSB induction is
phosphorylation of the C terminus of histone H2A on S129.
11
In higher eukary-
otes, this phosphorylation motif occurs not on the core H2A, but on the histone
variant H2AX
12
that constitutes approximately 10% of the total H2A.
13
Yeast
containing H2A that is not able to be phosphorylated have a defect in NHEJ and
are mildly sensitive to DNA-damaging agents,
11
while mouse cells lacking H2AX
are sensitive to IR and the animals are predisposed to cancer in the absence of
p53.
14-17
H2A phosphorylation is carried out by the damage-response phospha-
tidyl-3-OH-kinase-like kinases Tel1 and Mec1 in
S. cerevisiae
11
(homologous to
ataxia telangiectasia-mutated (ATM) and ATM- and Rad3-related (ATR), respec-
tively, inmammals) that additionally phosphorylate many other targets on SQ/TQ
motifs in response to damage.
18-20
H2A phosphorylation spreads over a large
region of
50 kb in budding yeast
21
or megabases in higher eukaryotes,
22
with
the levels of phosphorylation greatest at 3-5 kb from the break site, but low in the
1 kb immediately adjacent to the break.
23
Tel1, in conjunction with the nuclease MRX (Mre11-Rad50-Xrs2), is pri-
marily recruited to unprocessed DSBs. In contrast, Mec1 and its partner
protein Ddc1 are recruited to tracts of human replication protein A (RPA)-
bound single-stranded DNA. Single-stranded DNA is generated via processing
of DSBs through exonucleolytic resection by several nucleases including MRX
