Deubiquitinating enzymes and post-replication repair in Schizosaccharomyces pombe

Holmes, Rosalind Mary (2010) Deubiquitinating enzymes and post-replication repair in Schizosaccharomyces pombe. Doctoral thesis (DPhil), University of Sussex.

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Abstract

DNA damage is chronic, inevitable and extensive. Damage caused by UV irradiation can cause bulky DNA lesions that block replication forks. Postreplication repair (PRR) is a DNA damage tolerance mechanism, which enables the replication machinery to bypass DNA lesions. The PRR machinery is thought to be recruited by ubiquitination of the sliding clamp, PCNA. In human cells, the USP/UBP superfamily deubiquitinating enzyme (DUb) USP1 has been shown to remove ubiquitin from PCNA and hence acts as a PRR modulator. However, little is understood about the deubiquitination of PCNA or its regulation in yeast. The purpose of this study was to characterise the role of DUbs in yeast PRR. 24 DUbs were found to be encoded in the genome of Schizosaccharomyces pombe. No clear USP1 orthologue was found. A DUb deletion library was created and screened. A double mutant wherein two paralogous DUbs were deleted, ubp21D ubp22D, was found to exhibit sensitivity to UVC and increased PCNA ubiquitination. The ubp21D ubp22D strain was also found to be sensitive to a variety of DNA damaging agents and some spindle poisons. The double delete was epistatic with a mutant strain in which PCNA cannot be ubiquitinated. However, the genetic relationship with the enzymes that ubiquitinate PCNA was not so clear and a reduction in PCNA ubiquitination was not detected when either Ubp21 or Ubp22 was exogenously expressed. Ubp21 and Ubp22 also contain a meprin and TRAF homology (MATH) domain and a conserved DWGF motif in the MATH domain was found to be important for Ubp22 function. The human orthologue, HAUSPUSP7, stabilises the tumour suppressor p53 and is a highly characterised DUb. The Saccharomyces cerevisiae orthologue is Ubp15, but when this gene was deleted, only modest spindle poison sensitivity was detected. Determination of the precise functions of Ubp21 and Ubp22 in PRR requires further investigation.

Item Type: Thesis (Doctoral)
Schools and Departments: School of Life Sciences > Biochemistry
Subjects: Q Science > QD Chemistry
Q Science > QH Natural history > QH0301 Biology
Depositing User: Library Cataloguing
Date Deposited: 16 Jun 2010
Last Modified: 10 Aug 2015 13:06
URI: http://srodev.sussex.ac.uk/id/eprint/2378
Google Scholar:0 Citations

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