The histone H3-H4 chaperone Asf1 is involved in chromatin assembly (or 
disassembly), histone exchange, regulation of transcription, and chromatin 
silencing in several organisms. To investigate the essential functions of Asf1 
in Schizosaccharomyces pombe, asf1-ts mutants were constructed by random 
mutagenesis using PCR. One mutant (asf1-33(ts)) was mated with mutants in 77 
different kinase genes to identify synthetic lethal combinations. The asf1-33 
mutant required the DNA damage checkpoint factors Chk1 and Rad3 for its survival 
at the restrictive temperature. Chk1, but not Cds1, was phosphorylated in the 
asf1-33 mutant at the restrictive temperature, indicating that the DNA damage 
checkpoint was activated in the asf1-33 mutant. DNA damage occured in the 
asf1-33 mutant, with degradation of the chromosomal DNA observed through 
pulse-field gel electrophoresis and the formation of Rad22 foci. Sensitivity to 
micrococcal nuclease in the asf1-33 mutant was increased compared to the asf1(+) 
strain at the restrictive temperature, suggesting that asf1 mutations also 
caused a defect in overall chromatin structure. The Asf1-33 mutant protein was 
mislocalized and incapable of binding histones. Furthermore, histone H3 levels 
at the centromeric outer repeat region were decreased in the asf1-33 mutant and 
heterochromatin structure was impaired. Finally, sim3, which encodes a CenH3 
histone chaperone, was identified as a strong suppressor of the asf1-33 mutant. 
Taken together, these results clearly indicate that Asf1 plays an essential role 
in maintaining genomic stability in S. pombe.