Biology Reference
In-Depth Information
At this point in the activation cascade,
g
-H2AX has been activated and
repair factors are beginning to be recruited. This recruitment is facilitated by a
further decondensation of chromatin, which results in a number of chromatin
modifications becoming exposed. Dot1 trimethylates H3 at K79 and is impor-
tant in the recruitment of 53BP1, which activates checkpoint factors simulta-
neously to HRR.
9,10
This modification is considered constitutive, but becomes
unmasked during chromatin remodeling and functions as a binding site for
53BP1. Another methylation event that is constitutive, but important in HRR is
H4K20me. This modification is also unmasked during DNA repair and results
in the recruitment of multiple factors by their Tudor domains that recognize
H4K20me, which appears to be species specific.
11
There are a number of acetylation events that occur on H3 and H4 that
have been associated with the early events in HRR. These include H3 lysines
(K9, 14, 18, 23, and 27) and H4 lysines (K5, 8, 12, and 16). Multiple HATs have
been implicated in this process including GCN5, NuA4, and HAT1.
12,13
These
modifications increase near the DSB and are thought to be involved in loos-
ening the chromatin structure for proper repair. Interestingly, when the four
acetylated K residues on H4 are all mutated, DSB repair is abolished overall,
but their individual functions are not well established. H3 acetylation has been
most associated with HRR.
14
GCN5, an H3 HAT, has been well established in
the eviction of chromosomes during transcription and has more recently been
implicated in HRR, leading to the INO80-mediated chromosome eviction.
15,16
Specifically, H3 K14 and K23 appear to be the most critical events and are
stimulated by phosphorylation of H3 S10.
17
Mutation in these residues also
confers sensitivity to MMS, a chemotherapy which produces lesions primarily
repaired by HRR. Further, the competing pathway, NHEJ, protein DNA PKcs
has been reported to phosphorylate GCN5 in human cells and inactivate its
HAT domain,
18
indicating that GCN5 plays a pro-HRR role in DSB repair.
GCN5 also interacts with and coregulates BRCA1 depending on its HAT
activity, which further establishes it in the HRR pathway.
19
Protein ubiquitylation is an important process in cell biology rivaling the
significance of phosphorylation events. Ubiquitin requires a chain of events to
occur including several steps mediated by classes of proteins labeled E1, E2,
and E3 ligases. In short, ubiquitin is prepared by E1, ligated by E2, and
specificity is mediated by E3. There are more than 500 E3 ubiquitinating
enzymes and multiple mono- and polyubiquitin chain products. With the
complexity that has been described for this pathway, it has inevitably found a
role in chromatin modification that affects HRR. Not only is H2AX modified by
phosphorylation, it is also modified by ubiquitin dependent on the RNF8 (E3)
and UBC13 (E2) proteins, and this modification is present in colocalization
with
g
-H2AX.
20
This modification occurs downstream of
g
-H2AX and is depen-
dent on the binding of MDC1 to
g
-H2AX and the activity of p400, an SWI/SNF
