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

Abstract

One of the hallmarks of DNA damage is the rapid spreading of phosphorylated histone H2A (-H2AX) around a DNA double-strand break (DSB). In the budding yeast S. cerevisiae, nearly all H2A isoforms can be phosphorylated, either by Mec1 ATR or Tel1 ATM 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 ChIP-qPCR in order to uncover the mechanisms by which Mec1 ATR and Tel1 ATM propagate histone modifications across chromatin. With either kinase, -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 3D diffusive mechanism, whereas Tel1 undergoes directed motion along the chromatin.

Loop extrusion as a mechanism for DNA Double-Strand Breaks repair foci formation

“…Given that current estimates of cohesin-mediated loop extrusion suggest a rate of 0.5-2 kb per second in vitro 24,25 , such a mechanism would allow a rapid way to assemble DDR foci, with the entire megabase-sized chromatin domain being modified in about 10-30 min, which 9 fits with the observed rate of H2AX foci assembly 9 . This model would be in agreement with the recent finding that in yeast, the ATM ortholog Tel1 mediates H2A phosphorylation in a manner that agrees with a 1D sliding model rather than a 3D diffusion model 35 . Moreover, our…”