Determinants of late replication timing in fission yeast telomeres
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The process of DNA replication is highly regulated to ensure that DNA integrity is not compromised. Eukaryotic cells have multiple replication origins within their genome to initiate replication and ensure that all DNA is replicated within the time frame of S phase. It has proved to be very challenging to identify all of these replication origins. A factor that makes it more complicated to localize all replication origins is the temporal program that ensures that all replication origins do not fire at once; instead, their activation is spread out over the length of S phase. We wanted to identify the origin of replication in the telomere-associated sequences (TAS) of fission yeast so we could further study why it is a late-replicating origin. I found that the TAS origin is a strong, compound replication origin located in the region approximately 6-8 kb from the ends of chromosomes 1 and 2. The compound origin contains two independently functioning origins within a 2.4-kb region. Each of these can serve as an "autonomously replicating sequence (ARS)" on a plasmid, although the origin efficiencies of the individual ARS elements are reduced compared to the full compound origin. The primary determinant of late replication of the TAS origin appears to be cis -acting sequences located within a 5.6-kb region adjacent and telomere-proximal to the TAS origin. I showed that many sequences within the 5.6-kb region work together to prevent replication of the TAS until late S phase. We also wanted to study if the heterochromatic nature of the telomeres plays a role in their late replication. Absence of the Clr4 protein, which is necessary to establish Swi6-dependent heterochromatin, did not have any effect on TAS replication timing. The absence of important telomere maintenance proteins also did not change TAS replication timing. The lack of a chromosome end near the TAS replication origin also had no effect on replication timing. We did, however, find a partial acceleration of TAS origin firing in cds1&Delta; cells, indicating a role for checkpoint proteins in maintaining the replication timing program at fission yeast telomeres. This is consistent with microarray data showing that subtelomeres and telomeres can replicate in the presence of hydroxyurea in checkpoint-deficient fission yeast cells.