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in the acetylation and activation of ATM by Tip60.
51
Further, this would suggest
that the FATC domain of ATM interacts with MRN, as the FATC domain of
ATM was required for co-immunoprecipitation of both ATM and Tip60.
90
Determining the nature of the interactions between MRN, Tip60, and ATM,
and understanding how this complex functions to activate both ATM and Tip60
will require careful purification and biochemical analysis of these complexes.
These results suggest the following model to describe how the MRN
complex and Tip60 function together to promote the full activation of the
ATM kinase in response to DSBs. The initial step is the recruitment of the
MRN complex to the DSB, which is mediated by the interaction of the mre11
subunit of MRN with the broken DNA ends. Subsequently, ATM and Tip60 are
recruited to the MRN complex, concentrating these factors at the DSB.
Currently, it is unclear if ATM and Tip60 are recruited as a single ATM-
Tip60 complex, or if Tip60 and ATM bind separately to the MRN complex.
In either case, localization of ATM and Tip60 onto the chromatin allows the
chromodomain of Tip60 to interact with the nearby histone H3, which is
trimethylated on lysine 9 (H3K9me3). This interaction activates Tip60's acetyl-
transferase activity through allosteric regulation of the acetyltransferase do-
main, leading to acetylation of lysine 3016 of ATM and activation of ATM's
kinase activity. However, the relative contributions of the MRN complex and
Tip60 to the production of the fully active ATM kinase is currently unclear.
MRN could simply serve to recruit and concentrate the inactive ATM-Tip60
complex at DSBs, and thus stabilize the interaction between Tip60 and
H3K9me3 at DSBs. This would lead to activation of Tip60's acetyltransferase
activity and acetylation and activation of ATM kinase activity. However, purified
MRN can activate ATM in an
in vitro
biochemical system which appears to lack
Tip60,
38,49
indicating that MRN plays an active role in upregulating ATM
kinase activity. We favor the following model to account for activation of
ATM by both Tip60 and MRN. First, the binding of the MRN complex to
DNA may alter the conformation of the MRN complex, a process that may
involve the ATPase activity of the rad50 subunit.
38
This leads to recruitment of
ATM and Tip60 to MRN, either as a single ATM-Tip60 complex or as separate
proteins. Interaction between MRN and ATM then provides the initial activa-
tion of ATM's kinase activity. Tip60 activation through interaction with adjacent
H3K9me3 then leads to acetylation of the PRD domain of ATM. This acetyla-
tion then fixes ATM in the active conformation, allowing ATM to maintain
kinase activity even after dissociation from MRN. It is also possible that
interaction between MRN and ATM alters ATM's conformation to promote
direct acetylation by Tip60. In purified systems, although MRN activates ATM,
the absence of the normal mechanism for ATM inactivation (such as phospha-
tases and histone deacetylases
116
) may eliminate the normal
in vivo
require-
ment for acetylation of ATM by Tip60. In this model, both MRN and Tip60
