Investigating the role of Rts1 in DNA replication initiation

Understanding DNA replication initiation is essential to Background: understand the mis-regulation of replication seen in cancer and other human disorders. DNA replication initiates from DNA replication origins. In eukaryotes, replication is dependent on cell cycle kinases which function during S phase. Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK) act to phosphorylate the DNA helicase (composed of mini chromosome maintenance proteins: Mcm2-7) and firing factors to activate replication origins. It has recently been found that Rif1 can oppose DDK phosphorylation. Rif1 can recruit protein phosphatase 1 (PP1) to dephosphorylate MCM and restricts origin firing. In this study, we investigate a potential role for another phosphatase, protein phosphatase 2A (PP2A), in regulating DNA replication initiation. The PP2A regulatory subunit Rts1 was previously identified in a large-scale genomic screen to have a genetic interaction with (a DNA ORC2 replication licensing factor). Deletion of synthetically rescued the RTS1 temperature-sensitive (ts-) phenotype of mutants. ORC2 We deleted in multiple ts-replication factor Methods: RTS1 Saccharomyces strains, including . Dilution series assays were carried out to cerevisiae ORC2 compare qualitatively the growth of double mutant ts-replication factor ∆rts1 strains relative to the respective single mutant strains. No synthetic rescue of temperature-sensitivity was observed. Instead Results: we found an additive phenotype, indicating gene products function in separate biological processes. These findings are in agreement with a recent genomic screen which found that deletion in several ts-replication factor strains RTS1 led to increased temperature-sensitivity. We find no evidence that Rts1 is involved in the Conclusions: dephosphorylation of DNA replication initiation factors. Referee Status: AWAITING PEER REVIEW 06 Mar 2018, :23 (doi: ) First published: 3 10.12688/wellcomeopenres.13884.1 06 Mar 2018, :23 (doi: ) Latest published: 3 10.12688/wellcomeopenres.13884.1 v1 Page 1 of 11 Wellcome Open Research 2018, 3:23 Last updated: 07 MAR 2018