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bioRxiv · 10.1101/2022.10.09.511457

Live-Cell Tracking of γ-H2AX Kinetics Reveals the Distinct Modes of ATM and DNA-PK in Immediate Response to DNA Damage

Trakarnphornsombat, W.·Kimura, H.
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Abstract

DNA double-strand break (DSB) is a serious form of DNA damage that can cause genetic mutation. On the induction of DSBs, histone H2AX becomes phosphorylated by kinases, including ataxia telangiectasia-mutated (ATM), ataxia telangiectasia and Rad3-related (ATR), and DNA-dependent protein kinase (DNA-PK). Phosphorylated H2AX ({gamma}-H2AX) can be a platform to recruit DNA repair machinery. Here we analyzed the immediate early kinetics of {gamma}-H2AX upon laser-induced DNA damage in ATM-proficient and -deficient living cells by using fluorescently labeled antigen-binding fragments specific for {gamma}-H2AX. The accumulation kinetics of {gamma}-H2AX were similar in both ATM-proficient and -deficient cells. However, {gamma}-H2AX accumulation was delayed when the cells were treated with a DNA-PK inhibitor, suggesting that DNA-PK rapidly phosphorylates H2AX at DSB sites. Ku80, a DNA-PK subunit, diffused freely in the nucleus without DNA damage, whereas ATM repeatedly bound to and dissociated from chromatin. The H2AX phosphorylation activity of ATM, but not DNA-PK, depended on a histone H4K16 acetyltransferase, males absent on the first (MOF). These results suggest distinct actions of ATM and DNA-PK that plays a primary role in immediate early {gamma}-H2AX accumulation.

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Trakarnphornsombat, W., Kimura, H.. 2022-10-09. Live-Cell Tracking of γ-H2AX Kinetics Reveals the Distinct Modes of ATM and DNA-PK in Immediate Response to DNA Damage. https://doi.org/10.1101/2022.10.09.511457

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