Abstract
Ataxia telangiectasia mutated (ATM) is activated upon DNA double strand breaks (DSBs) and phosphorylates numerous DSB response proteins, including histone H2AX on serine 139 (Ser-139) to form γ-H2AX. Through interaction with MDC1, γ-H2AX promotes DSB repair by homologous recombination (HR). H2AX Ser-139 can also be phosphorylated by DNA-dependent protein kinase catalytic subunit and ataxia telangiectasia- and Rad3-related kinase. Thus, we tested whether ATM functions in HR, particularly that controlled by γ-H2AX, by comparing HR occurring at the euchromatic ROSA26 locus between mouse embryonic stem cells lacking either ATM, H2AX, or both. We show here that loss of ATM does not impair HR, including H2AX-dependent HR, but confers sensitivity to inhibition of poly(ADP-ribose) polymerases. Loss of ATM or H2AX has independent contributions to cellular sensitivity to ionizing radiation. The ATM-independent HR function of H2AX requires both Ser-139 phosphorylation and γ-H2AX/MDC1 interaction. Our data suggest that ATM is dispensable for HR, including that controlled by H2AX, in the context of euchromatin, excluding the implication of such an HR function in genomic instability, hypersensitivity to DNA damage, and poly(ADP-ribose) polymerase inhibition associated with ATM deficiency.
Highlights
Ataxia telangiectasia mutated (ATM) acts as a master controller of the double strand breaks (DSBs) response by phosphorylating numerous DSB response proteins, some of which, including histone H2AX, function in homologous recombination (HR)
Our data suggest that ATM is dispensable for HR, including that controlled by H2AX, in the context of euchromatin, excluding the implication of such an HR function in genomic instability, hypersensitivity to DNA damage, and poly(ADP-ribose) polymerase inhibition associated with ATM deficiency
Because at least three possible fractions of ␥-H2AX can be generated in response to DSBs, each by ATM, DNA-PKcs, and ATR, respectively, we investigated whether the ATM-dependent ␥-H2AX fraction is responsible for H2AX-dependent HR
Summary
ATM acts as a master controller of the DSB response by phosphorylating numerous DSB response proteins, some of which, including histone H2AX, function in homologous recombination (HR). Defective HR has been identified as a molecular mechanism causing genomic ionizing radiation; HU, hydroxyurea; ATM, ataxia telangiectasia mutated; DNA-PKcs, DNA-dependent protein kinase catalytic subunit; ATR, ataxia telangiectasia- and Rad3-related kinase; PARP, poly(ADP-ribose) polymerase; BRCT, BRCA1 C terminus; Gy, grays. Occurrence of chromatid breaks is at least in part due to inactivation of H2AX-dependent HR/sister chromatid recombination [10] In this regard, the difference in genomic abnormalities between ATMϪ/Ϫ cells and H2AXϪ/Ϫ cells implies that H2AX HR function may be independent of ATM. We found that ATM is dispensable for HR and not even required for HR controlled by ␥-H2AX
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