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Rad3 decorates critical chromosomal domains with gammaH2A to protect genome integrity during S-Phase in fission yeast.

Rozenzhak S, Mejía-Ramírez E, Williams JS, Schaffer L, Hammond JA, Head SR, Russell P - PLoS Genet. (2010)

Bottom Line: Unexpectedly, gammaH2A marks a diverse array of genomic features during S-phase, including natural replication fork barriers and a fork breakage site, retrotransposons, heterochromatin in the centromeres and telomeres, and ribosomal RNA (rDNA) repeats. gammaH2A formation at the centromeres and telomeres is associated with heterochromatin establishment by Clr4 histone methyltransferase.Brc1 C-terminal BRCT domain binding to gammaH2A is crucial in the absence of Rqh1(Sgs1), a RecQ DNA helicase required for rDNA maintenance whose human homologs are mutated in patients with Werner, Bloom, and Rothmund-Thomson syndromes that are characterized by cancer-predisposition or accelerated aging.We conclude that Rad3 phosphorylates histone H2A to mobilize Brc1 to critical genomic domains during S-phase, and this pathway functions in parallel with Rqh1 DNA helicase in maintaining genome integrity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, The Scripps Research Institute, La Jolla, California, United States of America.

ABSTRACT
Schizosaccharomyces pombe Rad3 checkpoint kinase and its human ortholog ATR are essential for maintaining genome integrity in cells treated with genotoxins that damage DNA or arrest replication forks. Rad3 and ATR also function during unperturbed growth, although the events triggering their activation and their critical functions are largely unknown. Here, we use ChIP-on-chip analysis to map genomic loci decorated by phosphorylated histone H2A (gammaH2A), a Rad3 substrate that establishes a chromatin-based recruitment platform for Crb2 and Brc1 DNA repair/checkpoint proteins. Unexpectedly, gammaH2A marks a diverse array of genomic features during S-phase, including natural replication fork barriers and a fork breakage site, retrotransposons, heterochromatin in the centromeres and telomeres, and ribosomal RNA (rDNA) repeats. gammaH2A formation at the centromeres and telomeres is associated with heterochromatin establishment by Clr4 histone methyltransferase. We show that gammaH2A domains recruit Brc1, a factor involved in repair of damaged replication forks. Brc1 C-terminal BRCT domain binding to gammaH2A is crucial in the absence of Rqh1(Sgs1), a RecQ DNA helicase required for rDNA maintenance whose human homologs are mutated in patients with Werner, Bloom, and Rothmund-Thomson syndromes that are characterized by cancer-predisposition or accelerated aging. We conclude that Rad3 phosphorylates histone H2A to mobilize Brc1 to critical genomic domains during S-phase, and this pathway functions in parallel with Rqh1 DNA helicase in maintaining genome integrity.

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γH2A forms at an ectopic RTS1 replication fork barrier.(A) Diagram illustrates position of RTS1 barrier located between two direct repeats of Ade6 alleles. Diagram based on [49]. (B) γH2A ChIP at sites surrounding the active or inactive RTS1 fork barrier. ChIP was performed on asynchronous cultures. A primer –3 kb from the RTS1 barrier in the MT locus was used as positive control.
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pgen-1001032-g004: γH2A forms at an ectopic RTS1 replication fork barrier.(A) Diagram illustrates position of RTS1 barrier located between two direct repeats of Ade6 alleles. Diagram based on [49]. (B) γH2A ChIP at sites surrounding the active or inactive RTS1 fork barrier. ChIP was performed on asynchronous cultures. A primer –3 kb from the RTS1 barrier in the MT locus was used as positive control.

Mentions: We next examined whether γH2A can form at an ectopic RTS1 fork barrier. We used a strain where RTS1 was inserted in the Ade6 locus, between two direct repeats (Figure 4A) [49]. This is a strong polar replication fork barrier and outside the MT locus fork stalling at RTS1 promotes recombination, which occurs without fork breakage, DSB formation, or checkpoint activation [49]. As a control we used an “inactive” strain in which the barrier is oriented in the opposite direction, thereby avoiding fork arrest (Figure 4A). ChIP analysis revealed a strong asymmetric enrichment of γH2A up to 5 kb away from the active RTS1 barrier (Figure 4B). The majority of γH2A was located on the right side of the barrier, where fork stalling occurs and recombination is initiated [49]. There was comparatively little γH2A on the left side of the barrier and no change in γH2A levels was detected when RTS1 orientation was reversed. A primer near RTS1 in the MT locus was used as a positive control for the experiment and showed similar levels of γH2A in both strains. These data show that fork stalling at a polar fork barrier triggers formation of an asymmetric γH2A domain and suggests that γH2A may mark recombination hotspots in the genome.


Rad3 decorates critical chromosomal domains with gammaH2A to protect genome integrity during S-Phase in fission yeast.

Rozenzhak S, Mejía-Ramírez E, Williams JS, Schaffer L, Hammond JA, Head SR, Russell P - PLoS Genet. (2010)

γH2A forms at an ectopic RTS1 replication fork barrier.(A) Diagram illustrates position of RTS1 barrier located between two direct repeats of Ade6 alleles. Diagram based on [49]. (B) γH2A ChIP at sites surrounding the active or inactive RTS1 fork barrier. ChIP was performed on asynchronous cultures. A primer –3 kb from the RTS1 barrier in the MT locus was used as positive control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2908685&req=5

pgen-1001032-g004: γH2A forms at an ectopic RTS1 replication fork barrier.(A) Diagram illustrates position of RTS1 barrier located between two direct repeats of Ade6 alleles. Diagram based on [49]. (B) γH2A ChIP at sites surrounding the active or inactive RTS1 fork barrier. ChIP was performed on asynchronous cultures. A primer –3 kb from the RTS1 barrier in the MT locus was used as positive control.
Mentions: We next examined whether γH2A can form at an ectopic RTS1 fork barrier. We used a strain where RTS1 was inserted in the Ade6 locus, between two direct repeats (Figure 4A) [49]. This is a strong polar replication fork barrier and outside the MT locus fork stalling at RTS1 promotes recombination, which occurs without fork breakage, DSB formation, or checkpoint activation [49]. As a control we used an “inactive” strain in which the barrier is oriented in the opposite direction, thereby avoiding fork arrest (Figure 4A). ChIP analysis revealed a strong asymmetric enrichment of γH2A up to 5 kb away from the active RTS1 barrier (Figure 4B). The majority of γH2A was located on the right side of the barrier, where fork stalling occurs and recombination is initiated [49]. There was comparatively little γH2A on the left side of the barrier and no change in γH2A levels was detected when RTS1 orientation was reversed. A primer near RTS1 in the MT locus was used as a positive control for the experiment and showed similar levels of γH2A in both strains. These data show that fork stalling at a polar fork barrier triggers formation of an asymmetric γH2A domain and suggests that γH2A may mark recombination hotspots in the genome.

Bottom Line: Unexpectedly, gammaH2A marks a diverse array of genomic features during S-phase, including natural replication fork barriers and a fork breakage site, retrotransposons, heterochromatin in the centromeres and telomeres, and ribosomal RNA (rDNA) repeats. gammaH2A formation at the centromeres and telomeres is associated with heterochromatin establishment by Clr4 histone methyltransferase.Brc1 C-terminal BRCT domain binding to gammaH2A is crucial in the absence of Rqh1(Sgs1), a RecQ DNA helicase required for rDNA maintenance whose human homologs are mutated in patients with Werner, Bloom, and Rothmund-Thomson syndromes that are characterized by cancer-predisposition or accelerated aging.We conclude that Rad3 phosphorylates histone H2A to mobilize Brc1 to critical genomic domains during S-phase, and this pathway functions in parallel with Rqh1 DNA helicase in maintaining genome integrity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, The Scripps Research Institute, La Jolla, California, United States of America.

ABSTRACT
Schizosaccharomyces pombe Rad3 checkpoint kinase and its human ortholog ATR are essential for maintaining genome integrity in cells treated with genotoxins that damage DNA or arrest replication forks. Rad3 and ATR also function during unperturbed growth, although the events triggering their activation and their critical functions are largely unknown. Here, we use ChIP-on-chip analysis to map genomic loci decorated by phosphorylated histone H2A (gammaH2A), a Rad3 substrate that establishes a chromatin-based recruitment platform for Crb2 and Brc1 DNA repair/checkpoint proteins. Unexpectedly, gammaH2A marks a diverse array of genomic features during S-phase, including natural replication fork barriers and a fork breakage site, retrotransposons, heterochromatin in the centromeres and telomeres, and ribosomal RNA (rDNA) repeats. gammaH2A formation at the centromeres and telomeres is associated with heterochromatin establishment by Clr4 histone methyltransferase. We show that gammaH2A domains recruit Brc1, a factor involved in repair of damaged replication forks. Brc1 C-terminal BRCT domain binding to gammaH2A is crucial in the absence of Rqh1(Sgs1), a RecQ DNA helicase required for rDNA maintenance whose human homologs are mutated in patients with Werner, Bloom, and Rothmund-Thomson syndromes that are characterized by cancer-predisposition or accelerated aging. We conclude that Rad3 phosphorylates histone H2A to mobilize Brc1 to critical genomic domains during S-phase, and this pathway functions in parallel with Rqh1 DNA helicase in maintaining genome integrity.

Show MeSH
Related in: MedlinePlus