Studies to determine the molecular mechanism of global genome nucleotide excision repair in S. cerevisiae

Zhou, Zheng 2007. Studies to determine the molecular mechanism of global genome nucleotide excision repair in S. cerevisiae. PhD Thesis, Cardiff University.

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Abstract

My thesis focuses on functions of Rad7/Radl6/AB1: complex (GG-NER complex) that is required for global genome repair (GG-NER), a subpathway of the nucleotide excision repair (NER) pathway, both in vivo and in vitro. Firstly, a putative DNA translocase activity of the GG-NER complex was investigated by using a triple helix strand displacement assay. Previous work in the lab showed that the complex could generate supercoiling in DNA. One way that DNA supercoiling can be induced is via DNA translocase activity. The GGR complex exhibits a similar level of DNA translocase activity to the SV-40 large T-antigen (a well characterised DNA translocase), indicating that the generation of superhelical torsion results from a translocase activity of the GG-NER complex. The activity is required during GGR to facilitate oligonucleotide excision. Secondly, I investigated a putative E3 ubiquitin ligase activity of a complex containing Rad7 and Radl6, and the stability of one of its substrates, Rad4. In response to UV irradiation, the native Rad4 protein which has a half-life of over three hours, is rapidly degraded by the ubiquitin-proteasome pathway. A novel Elongin-Cullin-Socs-box (ECS) type ubiquitin ligase complex, consisting of Rad7, Radl6, Cul3 and Elcl, was identified and was shown to be required for the UV-dependent ubiquitination and degradation of Rad4 in vivo. Furthermore, my data show that the SOCS box domain of Rad7 protein, which is required for the novel E3 ligase activity, was required for the UV-dependent ubiquitination and degradation of Rad4 protein and that Rad4 is a physiological target of this ligase. I showed that this Rad7 containing ubiquitin ligase ubiquitinates Rad4 protein in vitro and that a specific anti-Rad7 antibody inhibited this reaction, suggesting that the SOCS box protein Rad7 is an essential component of this novel ECS type ubiquitin ligase activity. When this ubiquitin ligase is inactive as is the case in the SOCS box mutated rad7 strain (psocs), no significant change in UV survival is observed compared to WT strain. However, when aArad23 mutation is combined with the psocs mutation a significant increase in UV sensitivity is observed in Arad23/psocs strain compared with Arad23 strain. This shows that the effect of the Rad7 containing ECS ligase on UV survival is predominantly observed in the absence of Rad23. I showed that the steady level of Rad4 after UV in psocs/Arad23 strain remains higher than in pRADllArad23 strain, but this does not rescue the UV sensitivity of psocs/Ara/23, indicating that the degradation of Rad4 protein does not correlate with UV survival. My results demonstrate that inducible NER is influenced by the Rad7 ECS ligase complex. Based on my data and other work from our group, it was revealed that ubiquitination of Rad4 in response to UV specifically regulates NER via a pathway that requires de novo protein synthesis, a pathway that is referred to as pathway II. Finally, preliminary experiments were designed and carried out to understand how the ubiquitination of Rad4 by the Rad7 ECS ligase functions in pathway II. In the absence of Rad23, the mRNA level of DDR2 is elevated. Furthermore, Rad23 binds to the promoter region of DDR2 in the absence of DNA damage. This suggests that Rad23 might regulate the transcription of DDR2 by directly binding to the regulatory elements of DDR2. Furthermore, the occupancy of Rad23 at the DDR2 promoter significantly decreased in Aelcl mutant cells, in psocs cells (an K3 ligase mutant), and after cells are exposed to UV, suggesting the possibility that Rad7 HCS ligase regulates a component of the transcriptional response to DNA damage.

Item Type:	Thesis (PhD)
Status:	Unpublished
Schools:	Medicine
Subjects:	R Medicine > RB Pathology
ISBN:	9781303182068
Funders:	ORS, Cardiff University School of Medicine
Date of First Compliant Deposit:	30 March 2016
Last Modified:	10 Jan 2018 06:13
URI:	https://orca.cardiff.ac.uk/id/eprint/55680

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