Analysis of the Role of the Cohesin Regulatory Proteins Pds5, Esco1, and Esco2 in DNA Replication
thesisposted on 10.01.2018, 12:55 by Lubinda Mukololo
Sister chromatid cohesion (SCC) is mediated by the cohesin complex whose core components are Smc1, Smc3, Scc1, and Scc3 (SA1 and SA2 in vertebrates) and is regulated by associated factors that include Pds5 (Pds5A and Pds5B in vertebrates) and the acetyltransferases Esco1 and Esco2. The correct establishment and maintenance of sister chromatid cohesion is important in safeguarding genome integrity. Previous studies have shown that Pds5 is important in the establishment and maintenance of SCC (in yeast and fungi) as well as sister chromatid resolution (in humans and Xenopus). In humans, both Esco1 and Esco2 acetylate Smc3 during DNA replication to establish SCC. Cohesin participates in DNA replication by modulating higher-order organisation of replication factories. Although disputed in Xenopus egg extracts, cohesin acetylation is reported to speed replication forks in human somatic cells. However, the mechanistic consequences of cohesin acetylation are still poorly understood and while Pds5’s role in SCC maintenance and resolution has been explored, no study to date has reported its role in DNA replication. Pilot studies (unpublished) in our lab have implicated Pds5 in DNA replication. In light of the foregoing, the aim of this project was to analyse the role of Pds5, Esco1, and Esco2 in DNA replication. Here, using an siRNA approach and DNA combing techniques in mammalian cells, I show that Pds5, Esco1, and Esco2 are functionally important in DNA replication. Depletion of either Esco1 or Esco2 results in precocious separation of sister chromatids, delay in DNA replication, apoptosis, or senescence. Depletion of Pds5 proteins is characterised by DNA damage which eventually activates the intra-S-phase DNA damage checkpoint that delays replication fork progression. Depletion of the anti-establishment complex, Pds5A and Wapl, rescues the defect in DNA replication observed when Pds5A is depleted alone. These results provide a novel insight into the role of Esco1, Esco2, and Pds5 in the regulation of DNA replication and suppression of aneuploidy in mammalian cells.