Fission Yeast Rad17 Associates with Chromatin in Response to Aberrant Genomic Structures

Abstract

Fission yeast checkpoint protein Rad17 is required for the DNA integrity checkpoint responses. A fraction of Rad17 is chromatin bound independent of the other checkpoint proteins throughout the cell cycle. Here we show that in response to DNA damage induced by either methyl methanesulfonate treatment or ionizing radiation, increased levels of Rad17 bind to chromatin. Following S-phase stall induced by hydroxyurea or a cdc22 mutation, the chromatin-bound Rad17 progressively dissociates from the chromatin. After S-phase arrest by hydroxyurea in cds1Δ or rad3Δ cells or by replication mutants, Rad17 remains chromatin bound. Rad17 is able to complex in vivo with an Rfc small subunit, Rfc2, but not with Rfc1. Furthermore, cells with rfc1Δ are checkpoint proficient, suggesting that Rfc1 does not have a role in checkpoint function. A checkpoint-defective mutant protein, Rad17(K118E), which has similar nuclear localization to that of the wild type, is unable to bind ATP and has reduced ability in chromatin binding. Mutant Rad17(K118E) protein also has reduced ability to complex with Rfc2, suggesting that Lys¹¹⁸ of Rad17 plays a role in Rad17-Rfc small-subunit complex formation and chromatin association. However, in the rad17.K118E mutant cells, Cds1 can be activated by hydroxyurea. Together, these results suggest that Rad17 binds to chromatin in response to an aberrant genomic structure generated from DNA damage, replication mutant arrest, or hydroxyurea arrest in the absence of Cds1. Rad17 is not required to bind chromatin when genomic structures are protected by hydroxyurea-activated Cds1. The possible checkpoint events induced by chromatin-bound Rad17 are discussed.

ACKNOWLEDGMENTS

We thank A. M. Carr for providing us with the myc-taggedrad17 strain, mutant rad17(K118E) strain, and plasmids pREP-myc-rad17 and pREP-myc-rad17(K118E). We also thank Hiroto Okayama for allowing Hiroyuki Tanaka to perform the rfc1 gene disruption experiment in his laboratory. We especially thank members of our laboratory for helpful discussion during the course of this work and R. E. Davis for his help in large-scale Rad17 protein purification.

This study was supported by grant CA54415 from the National Cancer Institute of the National Institutes of Health.