Schizosaccharomyces pombe Checkpoint Response to DNA Interstrand Cross-Links

Abstract

Drugs that produce covalent interstrand cross-links (ICLs) in DNA remain central to the treatment of cancer, but the cell cycle checkpoints activated by ICLs have received little attention. We have used the fission yeast, Schizosaccharomyces pombe, to elucidate the checkpoint responses to the ICL-inducing anticancer drugs nitrogen mustard and mitomycin C. First we confirmed that the repair pathways acting on ICLs in this yeast are similar to those in the main organisms studied to date (Escherichia coli, budding yeast, and mammalian cells), principally nucleotide excision repair and homologous recombination. We also identified and disrupted the S. pombe homologue of the Saccharomyces cerevisiae SNM1/PSO2 ICL repair gene and found that this activity is required for normal resistance to cross-linking agents, but not other forms of DNA damage. Survival and biochemical analysis indicated a key role for the “checkpoint Rad” family acting through the chk1-dependent DNA damage checkpoint in the ICL response. Rhp9-dependent phosphorylation of Chk1 correlates with G₂ arrest following ICL induction. In cells able to bypass the G₂ block, a second-cycle (S-phase) arrest was observed. Only a transient activation of the Cds1 DNA replication checkpoint factor occurs following ICL formation in wild-type cells, but this is increased and persists in G₂ arrest-deficient mutants. This likely reflects the fraction of cells escaping the G₂ damage checkpoint and arresting in the subsequent S phase due to ICL replication blocks. Disruption of cds1 confers increased resistance to ICLs, suggesting that this second-cycle S-phase arrest might be a lethal event.

ACKNOWLEDGMENTS

We thank J. Murray for kindly sharing unpublished strains.

This work was supported, in part, by EC grant FIGH-CT-1999-0010 (to A.M.C.) and was also partly supported by Cancer Research UK Programme grant SP2000/0402 (to J.A.H.). L.J.B. was supported by a Cancer Research UK studentship, and P.J.M. was supported by a Royal Society University Research fellowship.