Identification of two telomere-proximal fission yeast DNA replication origins constrained by nearby cis-acting sequences to replicate in late S phase.

Telomeres of the fission yeast,  Schizosaccharomyces pombe, are known to replicate in late S phase, but the reasons for this late replication are not fully understood. We have identified two closely-spaced DNA replication origins, 5.5 to 8 kb upstream from the telomere itself. These are the most telomere-proximal of all the replication origins in the fission yeast genome. When located by themselves in circular plasmids, these origins fired in early S phase, but if flanking sequences closer to the telomere were included in the circular plasmid, then replication was restrained to late S phase - except in cells lacking the replication-checkpoint kinase, Cds1. We conclude that checkpoint-dependent late replication of telomere-associated sequences is dependent on nearby cis-acting sequences, not on proximity to the physical end of a linear chromosome.

Checkpoint independence of most DNA replication origins in fission yeast.

BACKGROUND: In budding yeast, the replication checkpoint slows progress through S phase by inhibiting replication origin firing. In mammals, the replication checkpoint inhibits both origin firing and replication fork movement. To find out which strategy is employed in the fission yeast, Schizosaccharomyces pombe, we used microarrays to investigate the use of origins by wild-type and checkpoint-mutant strains in the presence of hydroxyurea (HU), which limits the pool of deoxyribonucleoside triphosphates (dNTPs) and activates the replication checkpoint. The checkpoint-mutant cells carried deletions either of rad3 (which encodes the fission yeast homologue of ATR) or cds1 (which encodes the fission yeast homologue of Chk2). RESULTS: Our microarray results proved to be largely consistent with those independently obtained and recently published by three other laboratories. However, we were able to reconcile differences between the previous studies regarding the extent to which fission yeast replication origins are affected by the replication checkpoint. We found (consistent with the three previous studies after appropriate interpretation) that, in surprising contrast to budding yeast, most fission yeast origins, including both early- and late-firing origins, are not significantly affected by checkpoint mutations during replication in the presence of HU. A few origins (approximately 3%) behaved like those in budding yeast: they replicated earlier in the checkpoint mutants than in wild type. These were located primarily in the heterochromatic subtelomeric regions of chromosomes 1 and 2. Indeed, the subtelomeric regions defined by the strongest checkpoint restraint correspond precisely to previously mapped subtelomeric heterochromatin. This observation implies that subtelomeric heterochromatin in fission yeast differs from heterochromatin at centromeres, in the mating type region, and in ribosomal DNA, since these regions replicated at least as efficiently in wild-type cells as in checkpoint-mutant cells. CONCLUSION: The fact that approximately 97% of fission yeast replication origins - both early and late - are not significantly affected by replication checkpoint mutations in HU-treated cells suggests that (i) most late-firing origins are restrained from firing in HU-treated cells by at least one checkpoint-independent mechanism, and (ii) checkpoint-dependent slowing of S phase in fission yeast when DNA is damaged may be accomplished primarily by the slowing of replication forks.