Yeast ATM and ATR kinases 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 (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.

Mapping yeast mitotic 5’ resection at base resolution reveals the sequence and positional dependence of nucleasesin vivo

“…Previous in vitro analysis of Mre11 activity on Ku-occluded dsDNA substrates revealed incisions at 35-45 and 55-65 bp from the end on 70 and 100 bp substrates, respectively (22,41), leading to the prevailing model wherein Mre11 creates a nick at a distance enforced by the Ku protein block (23). We observed a pattern of iterative peaks throughout this region consistent with the model and with the inference of a "stepwise" cleavage at somewhat regularly spaced positions Models cited above imply that Ku and/or Mre11 protein sizes are critical factors (22,23,41), but the pattern could be enforced by other proteins bound prior to damage such as histones or other proteins (42,43) and DNA repair proteins are can have sequence-specific properties (44,45). We addressed these issues using 3 bp deletions in the ILV1-PR sequence.…”

“…A central question is what determines the pattern of cleavage at mitotic DSBs in vivo . Models cited above imply that Ku and/or Mre11 protein sizes are critical factors (22, 23, 41), but the pattern could be enforced by other proteins bound prior to damage such as histones or other proteins (42, 43) and DNA repair proteins are can have sequence-specific properties (44, 45). We addressed these issues using 3 bp deletions in the ILV1 -PR sequence.…”

A Tale of Ice and Fire: The Dual Role for 17β-Estradiol in Balancing DNA Damage and Genome Integrity

“…ATM kinase is recruited and activated in the presence of a DSB thanks to the MRE11/RAD50/NBS1 (MRN) sensor complex [ 54 , 55 , 56 ]. ATR is activated together with its partner ATRIP in response to a single-strand break (SSB) thanks to the presence of the replication protein A (RPA) sensor [ 57 , 58 , 59 , 60 , 61 ]. DNA-PKcs is activated by DSB and is activated by Ku [ 48 , 62 ].…”

“…In addition, cyclin D1 is recruited by E2 to contribute to the regulation of the DDR pathway via an extra-nuclear mechanism [ 61 ]. Thus, ERα-cyclin D1 binding at the cytoplasmic membrane augments AKT phosphorylation (Ser473) and γH2AX foci formation.…”

DNA damage-induced phosphorylation of histone H2A at serine 15 is linked to DNA end resection

“…Yeast Lcd1/Ddc2 (mammalian ATRIP) is responsible for the recruitment of Mec1 to RPA-coated single stranded DNA (ssDNA) produced during end resection (34). Mec1 has been shown to be involved in spreading of g-H2A in trans onto adjacent undamaged chromosomes in close physical proximity (10,35). Deletion of LCD1 is known to incur loss of Mec1 recruitment to RPA-coated ssDNA and therefore Mec1-dependent cell signaling in response to damage (36,37).…”