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Continued DNA synthesis in replication checkpoint mutants leads to fork collapse.

ABSTRACT: Hydroxyurea (HU) treatment activates the intra-S phase checkpoint proteins Cds1 and Mrc1 to prevent replication fork collapse. We found that prolonged DNA synthesis occurs in cds1? and mrc1? checkpoint mutants in the presence of HU and continues after release. This is coincident with increased DNA damage measured by phosphorylated histone H2A in whole cells during release. High-resolution live-cell imaging shows that mutants first accumulate extensive replication protein A (RPA) foci, followed by increased Rad52. Both DNA synthesis and RPA accumulation require the MCM helicase. We propose that a replication fork "collapse point" in HU-treated cells describes the point at which accumulated DNA damage and instability at individual forks prevent further replication. After this point, cds1? and mrc1? forks cannot complete genome replication. These observations establish replication fork collapse as a dynamic process that continues after release from HU block.

SUBMITTER: Sabatinos SA

PROVIDER: S-EPMC3510540 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Continued DNA synthesis in replication checkpoint mutants leads to fork collapse.

Sabatinos Sarah A SA Green Marc D MD Forsburg Susan L SL

Molecular and cellular biology 20121008 24

Hydroxyurea (HU) treatment activates the intra-S phase checkpoint proteins Cds1 and Mrc1 to prevent replication fork collapse. We found that prolonged DNA synthesis occurs in cds1Δ and mrc1Δ checkpoint mutants in the presence of HU and continues after release. This is coincident with increased DNA damage measured by phosphorylated histone H2A in whole cells during release. High-resolution live-cell imaging shows that mutants first accumulate extensive replication protein A (RPA) foci, followed b ...[more]

PMID: 23045396

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