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The Rad24 checkpoint protein of Saccharomyces cerevisiae: A complex problem

Green, Catherine Mary; (2000) The Rad24 checkpoint protein of Saccharomyces cerevisiae: A complex problem. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Rad24 functions in the DNA damage-dependent checkpoint pathway of Saccharomyces cerevisiae. Polyclonal antibodies were raised against Rad24 and other components of this pathway in order to investigate their biochemical functions. Studies demonstrated that Rad24, Mec3 or Rad17 did not interact with each other and did not appear to be modified after DNA damage treatment or during the cell cycle. Analysis of Rad24 in whole cell extracts demonstrated that its mass was considerably greater than its predicted molecular weight, suggesting that Rad24 is a component of a protein complex. A protocol was developed to purify the Rad24 complex to homogeneity. In addition to Rad24, the complex included polypeptides of 40kDa and 35kDa. The 40kDa species was found to contain Rfc2 and Rfc3 by mass spectrometry. Rfc2 and Rfc3 are subunits of Replication Factor C (RFC), a five subunit protein which is required for the loading of polymerases onto DNA during replication and repair. We hypothesised that other RFC subunits, all of which share sequence homologies with Rad24, might also be components of the Rad24 complex. Reciprocal co-immunoprecipitation studies were performed using extracts prepared from strains constructed containing epitope tagged RFC genes. These experiments showed that the small RFC proteins, Rfc2, Rfc3, Rfc4 and Rfc5 interact with Rad24, whereas the Rfc1 subunit does not. We suggest that this RFC-like Rad24 complex may function as a structure-specific sensor in the DNA damage checkpoint pathway. In order to address this hypothesis, conditional mutants of small Rfc subunits were tested for intact DNA damage responses outside of S phase. The biochemical activities of the Rad24 complex were investigated using a variety of techniques including gel mobility shift and ATP hydrolysis assays.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: The Rad24 checkpoint protein of Saccharomyces cerevisiae: A complex problem
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest
Keywords: Pure sciences; Polyclonal antibodies
URI: https://discovery.ucl.ac.uk/id/eprint/10097282
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