Abstract:
Hsk1 is a protein kinase found in Schizosaccharomyces pombe and is known to have active roles in DNA replication. It is also found to associate with a regulatory subunit called Dfp1 to form a complex equivalent to Cdc7/Dbf4 in eukaryotes. During replication, this kinase has been shown to play a crucial role in phosphorylation MCM proteins. Recent studies have shown Hsk1-Dfp1 complex is now known to interact with the heterochromatin protein 1 homolog, Swi6, to promote formation of heterochromatin and cohesion especially at the centromeres. Disruption of this recruitment of Dfp1 by Swi6 to the pericentromeric region causes delay in mitosis. The homolog of this complex in S. cerevisiae, Cdc7-Dbf4 Kinase, is further attributed with roles in phosphorylation of cohesin proteins such as Rec8 in order to restrict the cleavage of cohesins at centromeres specifically to meiosis II. The same is also known to phosphorylate kinetochore proteins such as Ctf19. This process is essential for recruitment of the cohesin loader complex to the chromosome and thereby cohesin loading. Similar interactions with Rec8 have been shown to occur in S. pombe. But, interaction of Dfp1 with other cohesin subunits and cohesin loader complex is still largely unknown in S. pombe. During this study, we primarily planned to look at the role of Hsk1-Dfp1 in cohesin loading at the centromeres by attempting to characterize the interactions between the regulatory subunit Dfp1 and different centromeric proteins in S. pombe. We also aimed to characterize the mutants hsk1-1312 and two truncation mutants dfp1(1-376) and dfp1(1-494) to understand whether their roles in cohesin loading is critical in chromosomal segregation and viability. Forced localization of cohesins to the centromere was used as a strategy to test this hypothesis and here we discuss our observations and results.
