In S. cerevisiae initiation of replication occurs from discrete sites in the genome, known as origins and these display a characteristic temporal profile of activation during S phase of the cell cycle. The genomic context of origins has been demonstrated to be important to determine the time of firing, more specifically histone acetylation levels surrounding origins can influence their activation time. How increased acetylation is translated into earlier firing of specific origins is currently unknown. Bromodomains are known to bind acetylated histones in vivo. The bromodomain-containing Yta7p has been identified in a complex with various remodelers of chromatin and subunits of DNA polymerase ǫ. It is also a target of cell cycle and checkpoint kinases. Therefore, Yta7p makes an excellent candidate to bind acetylated histones surrounding replication origins and affect an alteration in the chromatin structure that could influence time of firing. Deletion of the histone deacetylase RPD3 results in a rapid S phase phenotype due to increased histone acetylation at “late-firing” origins. Increased acetylation at “late” origins leads to an advance in the time of firing of those specific origins. The aim of this study was to investigate the hypothesis that the bromodomain-containing protein Yta7p binds to histones with increased acetylation near to replication origins and subsequently influences origin firing. Hence, deletion of YTA7 would abolish the rapid S phase of a ∆rpd3 strain. Indeed the S phase of the ∆rpd3∆yta7 strain was reverted to WT duration. A role for Yta7p in DNA replication is also inferred by two additional lines of evidence presented in this thesis. Synthetic growth defects are evident when YTA7 and RPD3 deletion is combined with mutation of a third replication protein. In addition, ∆rpd3∆yta7 mutants are sensitive to HU, which is a phenotype shared by many strains with deletions in genes that encode proteins involved in DNA replication. Evidence to support a direct role of Yta7p in DNA replication events is provided by identification of an S phase specific binding of Yta7p to replication origins. Moreover, levels of Yta7p bound to early-firing origins are increased compared with their later-firing counterparts. Levels of Yta7p that are bound to “late-firing” origins are only increased in conditions of RPD3 deletion, where the resulting increase in histone acetylation at the “late-firing” origins is associated with advanced time of firing. Time of Yta7p binding at these “late” origins is also advanced concomitantly. This data supports the hypothesis that Yta7p provides a functional link between histone acetylation and time of origin activation. In searching for a specific replication linked function of Yta7p it was observed that recruitment of the FACT subunit Spt16p to replication origins was increased in conditions of YTA7 deletion. A second function for Yta7p in the S phase checkpoint was also demonstrated and the two roles of Yta7p, in DNA replication and S phase checkpoint, were separated depending upon their requirement for the bromodomain. The data produced in this thesis adds to our knowledge of DNA replication events and highlights the importance of histone modifications and chromatin remodeling to the replication field. This thesis describes the direct involvement of a protein, which was previously unassociated, with DNA replication and S phase checkpoint function and provides good ground work for future investigation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:563062 |
| Date | January 2010 |
| Creators | Curley, Rebecca |
| Contributors | Vogelauer, Maria. : Harrington, Lea |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/4811 |
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