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RESEARCH PAPER

Replisome instability, fork collapse, and gross chromosomal rearrangements arise synergistically from Mec1 kinase and RecQ helicase mutations

¹ Frontiers in Genetics NCCR Program, University of Geneva, CH-1211 Geneva 4, Switzerland; ² Centro de Biología Molecular "Severo Ochoa," Universidad Autónoma de Madrid/CSIC, Cantoblanco, 28049-Madrid, Spain; ³ Friedrich Miescher Institute for Biomedical Research, CH-4058 Basel, Switzerland

The yeast checkpoint kinases Mec1 and Rad53 are required for genomic stability in the presence of replicative stress. When replication forks stall, the stable maintenance of replisome components requires the ATR kinase Mec1/Ddc2 and the RecQ helicase Sgs1. It was unclear whether either Mec1 or Sgs1 action requires the checkpoint effector kinase, Rad53. By combining sgs1 with checkpoint-deficient alleles, we can now distinguish the role of Mec1 at stalled forks from that of Rad53. We show that the S-phase-specific mec1-100 allele, like the sgs1 mutation, partially destabilizes DNA polymerases at stalled forks, yet combining the mec1-100 and sgs1 mutations leads to complete disassociation of the replisome, loss of RPA, irreversible termination of nucleotide incorporation, and compromised recovery from hydroxyurea (HU) arrest. These events coincide with a dramatic increase in both spontaneous and HU-induced chromosomal rearrangements. Importantly, in sgs1 cells, RPA levels at stalled forks do not change, although Ddc2 recruitment is compromised, explaining the partial Sgs1 and Mec1 interdependence. Loss of Rad53 kinase, on the other hand, does not affect the levels of DNA polymerases at arrested forks, but leads to MCM protein dissociation. Finally, confirming its unique role during replicative stress, Mec1, and not Tel1, is shown to modify fork-associated histone H2A.

[Keywords: Replicative stress; checkpoint; DNA polymerases; Mec1; Sgs1; chromosome instability]

Received August 3, 2005; revised version accepted October 24, 2005.

Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/gad.361805.

⁴ Present address: Department of Molecular Biology, Aarhus University, DK-8000 Aarhus C, Denmark.

⁵ Corresponding author.

E-MAIL susan.gasser{at}fmi.ch; FAX 41-61-697-39-76.

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