Yeast ATM and ATR kinases use different mechanisms to spread histone H2A phosphorylation around a DNA double-strand break

Significance Creation of a chromosomal double-strand break (DSB) is rapidly accompanied by extensive phosphorylation of yeast histone H2A isoform (H2AX in mammals) by Mec1 (ATR) and Tel1 (ATM) protein kinases. This phosphorylation, termed γ-H2AX, spreads 50 kb on either of the DSB, but how this phosphorylation is propagated is poorly understood. We have monitored the kinetics and extent of spreading individually for Mec1 and Tel1 and find that the patterns of spreading are significantly different for the two kinases. By comparing these experimental data to mathematical models of spreading, either by one-dimensional (1D) or 3D diffusion, we report that Bayesian model selections suggest Tel1 acts by directed motion along a chromatin fiber, whereas Mec1 primarily uses a 3D mode of propagation.
One of the hallmarks of DNA damage is the rapid spreading of phosphorylated histone H2A (γ-H2AX) around a DNA double-strand break (DSB). In the budding yeast Saccharomyces cerevisiae, nearly all H2A isoforms can be phosphorylated, either by Mec1ATR or Tel1ATM checkpoint kinases. We induced a site-specific DSB with HO endonuclease at the MAT locus on chromosome III and monitored the formation of γ-H2AX by chromatin immunoprecipitation (ChIP)-qPCR in order to uncover the mechanisms by which Mec1ATR and Tel1ATM propagate histone modifications across chromatin. With either kinase, γ-H2AX spreads as far as ∼50 kb on both sides of the lesion within 1 h; but the kinetics and distribution of modification around the DSB are significantly different. The total accumulation of phosphorylation is reduced by about half when either of the two H2A genes is mutated to the nonphosphorylatable S129A allele. Mec1 activity is limited by the abundance of its ATRIP partner, Ddc2. Moreover, Mec1 is more efficient than Tel1 at phosphorylating chromatin in trans—at distant undamaged sites that are brought into physical proximity to the DSB. We compared experimental data to mathematical models of spreading mechanisms to determine whether the kinases search for target nucleosomes by primarily moving in three dimensions through the nucleoplasm or in one dimension along the chromatin. Bayesian model selection indicates that Mec1 primarily uses a three-dimensional diffusive mechanism, whereas Tel1 undergoes directed motion along the chromatin.