Biology Reference
In-Depth Information
53BP1.
3
Interestingly, an AA substitution for glutamic acid, mimicking phos-
phorylation, does not induce localization of repair factors indicative that
g
-H2AX is only one step in a more complicated process.
4
No text on
g
-H2AX in the context of chromatin would be complete without
a discussion on
g
-H2AX's role in the relaxation of heterochromatin. It is still up
for debate whether the phosphorylation of H2AX induces chromatin relaxation,
or whether relaxed chromatin is more likely to be marked by phosphorylation.
The first model is that the phosphorylation event following damage recruits
proteins that relax heterochromatin in preparation for repair. This is supported
by the general finding that
g
-H2AX foci are rarely seen in heterochromatin,
indicating that the structure is relaxed rapidly after phosphorylation has oc-
curred, as has been reported around a DSB. Second, it could be that hetero-
chromatin is structurally less likely to be marked. As heterochromatin is more
compacted, it yields less access to kinases, less phosphorylation, and further, is
less likely to be damaged by oxidative damage produced during IR or by
chemotherapy.
5
In either of the current models,
g
-H2AX has become a marker for the
presence and/or location of DSBs within the cell. It is a commonly used
antibody for determining the presence of DSBs using multiple laboratory
techniques including ChIP, Western blotting, and immunofluorescence. It
has been established that there are near 1 to 1 ratios (0.7-0.9 depending
on cell type) of DNA DSBs to
g
-H2AX nuclear foci in mammalian cells,
which is especially useful in localizing other factors to sites of DNA DSBs.
Of note,
g
-H2AX is phosphorylated in most DNA DSBs including those
produced during DNA replication and those frank DSBs that will be repaired
via nonhomologous end-joining (NHEJ), which is discussed separately in this
chapter.
The other H2A variant, H2AZ, is less well defined in DNA repair. In
transcription, H2AZ is inserted in most RNA polymerase II promoters to
allow easier eviction. Its role in repair is thought to be similar to this. It has
been shown in yeast to be inserted by SWR1 around DSBs depending on
sumoylation at K126 and K133 in the C-terminal tail, and this relocalization is
important for further steps in the HRR process including RAD51 function.
6
This process likely enhances chromatin eviction in preparation for HRR and
has been shown to occur during the initial steps in the process to influence
access to chromatin.
The initial steps in the DSB pathway involve Tip60 (Tat-interacting protein,
or NuA4 in yeast), MRN, and ATM, which have been more recently elucidated
to shed light on the events leading to
g
-H2AX production.
7
The current model
begins with undamaged, normal chromatin compacted and stabilized by the
heterochromatin protein 1 (HP1) and KRAB-associated protein (KAP-1).
These proteins sit on heterochromatin and maintain the condensed state.
