Genome-wide localization of γH2A during DNA replication.
(A) Genome-wide (ChIP-on-chip) distribution of γH2A during S phase. Schematics represent the three S. pombe chromosomes with key structural features (top). Tel- telomere; cen-centromere; MT-mating-type locus; rDNA-ribosomal DNA. Enrichment of γH2A is displayed as MAT scores (y-axis). Chromosome coordinates (x-axis, in megabases, (Mb)) downloaded from the S.pombe Genome Project (Sanger Center: www.sanger.ac.uk/Projects/S_pombe). (B) (left) γH2A formation occurs specifically during S phase. ChIP-qPCR timecourse analysis of γH2A enrichment at the indicated sites was performed by synchronizing cells using cdc25-22 block and release, and ChIP samples were collected every 30 min. Cell cycle progression was monitored by septation index. (right) Western blots comparing levels of γH2A in cdc25-22 wild type (Cds1+) and cdc25-22 cds1Δ cells released into 12 mM HU from G2 arrest. As an untreated control cdc25-22 wild type cells were released from G2 in the absence of HU. (C) Rad3 is the main kinase that phosphorylates H2A during unperturbed S phase. γH2AChIP-qPCR in wild type, rad3Δ, or tel1Δ cells synchronized as in (B), samples were collected in G2 and S phase. Primers: MT–5 kb from DSB in MT-locus; tel-subtelomere 1; cen-dh – centromere dh repeats; cnt – centromere 1 core; rDNA- 35S ribosomal DNA gene.