Disruption of Mechanisms That Prevent Rereplication Triggers a DNA Damage Response

Abstract

Eukaryotes replicate DNA once and only once per cell cycle due to multiple, partially overlapping mechanisms efficiently preventing reinitiation. The consequences of reinitiation are unknown. Here we show that the induction of rereplication by mutations in components of the prereplicative complex (origin recognition complex [ORC], Cdc6, and minichromosome maintenance proteins) causes a cell cycle arrest with activated Rad53, a large-budded morphology, and an undivided nucleus. Combining a mutation disrupting the Clb5-Orc6 interaction (ORC6-rxl) and a mutation stabilizing Cdc6 (CDC6ΔNT) causes a cell cycle delay with a similar phenotype, although this background is only partially compromised for rereplication control and does not exhibit overreplication detectable by fluorescence-activated cell sorting. We conducted a systematic screen that identified genetic requirements for the viability of these cells. ORC6-rxl CDC6ΔNT cells depend heavily on genes required for the DNA damage response and for double-strand-break repair by homologous recombination. Our results implicate an Mre11-Mec1-dependent pathway in limiting the extent of rereplication.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/.

ACKNOWLEDGMENTS

We thank Alison North for help with DeltaVison microscopy. V.A. thanks Michael P. Rout for providing laboratory space during the initial part of this work. We thank Lea Schroeder and Veronica Campbell for technical assistance and John Petrini (Memorial Sloan-Kettering Cancer Research Institute), Xiaolan Zhao (The Rockefeller University), John Diffley (Clare Hall Laboratories, Cancer Research United Kingdom), Stephen Elledge (Harvard University, Cambridge, MA), and Steve Brill (Rutgers University) for strains. We also thank Stephen Elledge for the gift of anti-Rad53 serum. We thank the reviewers for their careful readings of the manuscript and their useful comments.

Funding was provided by PHS grant GM047238 to F. C. A.E.I. was funded by the Charles H. Revson Foundation.