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Title: A strategy to isolate, in vivo, a eukaryotic replication origin for the analysis of its asscoiated proteins
Author: Mariotti, Francesca Romana
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2011
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In all organisms the DNA content is precisely and efficiently replicated. DNA replication starts at specific regions called replication origins, where a pre- Replicative Complex (pre-RC) is assembled at the end of the G2/M phase, maldng chromatin competent for replication. At the transition between G1 and S phases the activation of two conserved protein kinases triggers the transition from pre-RC into the active form, the Replicative Complex (RC). Replication origins are not fired at the same time during S phase, thus we can define origins as 'early' and 'late' according to their time of activation. During the past years our knowledge about DNA replication has improved due to the identification of novel proteins. Despite a large body of work, it remains possible that additional factors remain unidentified. The discovery of new proteins by genetic means is difficult due to the presence of redundant mechanisms and to their requirement for cell survival. Thus the aim of my project is to develop an unbiased assay to study, in Saccharomyces cerevisiae, the proteome of a single eukaryotic replication origin and identify novel proteins involved in DNA replication. Because the chromosomal environment profoundly influences the behaviour of replication origins (such as timing, efficiency), it is therefore of interest to analyse the proteomics of replication origins in their endogenous context. Taking advantage of the pSRl recombination system, I constructed two plasmids (pLTl/RsiteA and pLT2/RsiteB) that led to the integration of recombination sites into the yeast genome and the excision in vivo of the late replication origin ARS1413. These plasmids, allowing the excision of any flanking region, represent important genetic tools. The resulting episome has been isolated from the genome by density gradient purification in order to analyze by mass spectrometry the protein associated with the specific replication origin. This technique can be used to study the proteins involved in different steps of DNA replication. In fact cells arrested in G1 with a-factor will provide the proteome of the pre-RC while cells treated with HU will represent the proteome of late origins that are inhibited by the S-phase checkpoint.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available