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.


CONCLUSIONS
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.
Mickle
Katie L
KL
Department of Microbiology and Molecular Genetics, SUNY at Stony Brook, Stony Brook, New York 11794-5222, USA. katie.mickle@gmail.com
Ramanathan
Sunita
S
Rosebrock
Adam
A
Oliva
Anna
A
Chaudari
Amna
A
Yompakdee
Chulee
C
Scott
Donna
D
Leatherwood
Janet
J
Huberman
Joel A
JA
D018797:Cell Cycle Proteins / Q000235:genetics; D064447:Checkpoint Kinase 2; D002875:Chromosomes / Q000235:genetics; D004261:DNA Replication / Q000187:drug effects / Q000502:physiology*; D004271:DNA, Fungal / Q000235:genetics / Q000502:physiology*; D006918:Hydroxyurea / Q000494:pharmacology; D046228:Microarray Analysis / Q000379:methods; D009154:Mutation; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D018741:Replication Origin / Q000235:genetics*; D012568:Schizosaccharomyces / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics
D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D019384:Nucleic Acid Synthesis Inhibitors; D029702:Schizosaccharomyces pombe Proteins; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C578636:Cds1 protein, S pombe; D017346:Protein Serine-Threonine Kinases; C456840:rad3 protein, S pombe; D006918:Hydroxyurea