Mechanistic studies of DNA repair and genome instability

Thesis

The accurate repair of DNA damage is essential for protecting cells from undergoing cell death or accumulating mutations. The multi-topical work presented within this thesis focuses on the mechanistic study of DNA repair and sources of genome instability.

Interstrand crosslinks (ICLs) are toxic DNA lesions that block the essential cellular processes of transcription and replication. The mechanisms of ICL repair pathways in non-dividing cells are poorly characterized. This study aims to determine the mechanisms for a transcription- associated ICL repair pathway. After developing an in vitro biochemical system to study ICL repair, it is demonstrated that the CSB transcription-coupled repair factor can directly stimulate the SNM1A exonuclease in the unhooking of ICLs. A novel functional stimulatory interaction between the XPF-ERCC1 endonuclease and the SNM1A exonuclease is also identified. The experiments described here suggest that transcription-associated repair of ICLs may result in formation of toxic DSBs that are repaired by non-homologous end joining.

It was recently identified that cancer cells with microsatellite instability (MSI) undergo cell death after genetic knockdown of the Werner helicase (WRN), though the mechanism for this is unknown. We provide evidence that depletion of WRN in MSI cells generates DNA double- strand breaks (DSBs) at (TA)n microsatellite repeats. In MSI cancer cells, (TA)n microsatellite repeats undergo expansion and form stable non-B DNA cruciform structures. If these structures are not unwound by the WRN helicase, they are cleaved by the MUS81-EME1 nuclease, resulting in DSB formation across the genome. This study identifies expanded (TA)n repeats as a biomarker for MSI cells’ synthetic lethal dependence on WRN and supports the concept of developing WRN inhibitors for the treatment of MSI cancers.

Neurons are long-lived, post-mitotic cells that are believed to face high rates of endogenous DNA damage. Surprisingly little is known about the causes of endogenous DNA damage in neurons and where in the genome the damage occurs. We demonstrate that DNA single- strand breaks (SSBs) occur at neuronal enhancers. These SSBs are largely repaired through PARP1, XRCC1, and Pol β-dependent mechanisms. The sites of these breaks overlap with sites of TET-mediated oxidation of 5-methylcytosine, suggesting that demethylation of DNA could be the source of endogenous SSB formation at neuronal enhancers. This work highlights a novel research technique called S1-END-seq for the genomic mapping of DNA SSBs.

Authors: Nathan, WJ

• DOI: 10.5287/ora-1amapaoap
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: MUS81-EME1; DNA repair; XPF; neurodegeneration; cruciform; neurons; cockayne syndrome; SNM1A; genome instability; interstrand crosslink; single strand break; transcription; non-dividing cell; WRN; microsatellite instability; cancer; demethylation
• Subjects: Cancer; Aging; Nervous system--Degeneration; DNA repair; Chemotherapy; Biochemistry; DNA damage