Activation of the DNA Damage Checkpoint in Yeast Lacking the Histone Chaperone Anti-Silencing Function 1

Abstract

The packaging of the eukaryotic genome into chromatin is likely to be important for the maintenance of genomic integrity. Chromatin structures are assembled onto newly synthesized DNA by the action of chromatin assembly factors, including anti-silencing function 1 (ASF1). To investigate the role of chromatin structure in the maintenance of genomic integrity, we examined budding yeast lacking the histone chaperone Asf1p. We found that yeast lacking Asf1p accumulate in metaphase of the cell cycle due to activation of the DNA damage checkpoint. Furthermore, yeast lacking Asf1p are highly sensitive to mutations in DNA polymerase alpha and to DNA replicational stresses. Although yeast lacking Asf1p do complete DNA replication, they have greatly elevated rates of DNA damage occurring during DNA replication, as indicated by spontaneous Ddc2p-green fluorescent protein foci. The presence of elevated levels of spontaneous DNA damage in asf1 mutants is due to increased DNA damage, rather than the failure to repair double-strand DNA breaks, because asf1 mutants are fully functional for double-strand DNA repair. Our data indicate that the altered chromatin structure in asf1 mutants leads to elevated rates of spontaneous recombination, mutation, and DNA damage foci formation arising during DNA replication, which in turn activates cell cycle checkpoints that respond to DNA damage.

We thank Paul Megee for critical reading of the manuscript. We are very grateful to Jim Haber, Rohinton Kamakaka, Paul Megee, Jocelyn Krebs, Michael Fasullo, and Bob Sclafani for yeast strains; David Toczyski for the Ddc2p-GFP plasmid; Noel Lowndes for antibodies to Rad53 and Rad9; and Steven Jackson and Jocelyn Krebs for antibodies to phosphorylated H2A. We thank Miguel Ferreria for stimulating discussions and help and advice with pulsed-field gel electrophoresis and Michelle Pham, Josh Carson, and Jack Milwid for technical assistance. We are particularly grateful to the University of Colorado Cancer Center Flow Cytometry Facility for flow cytometry analyses.

This study was supported by an NIH award CA95641-01 to J.K.T. J.K.T. is a scholar of the Leukemia and Lymphoma Society. C.J.R. was supported by a predoctoral training grant in molecular biology NIH T32GM 08730.