Identification of novel players implicated in replication-dependent and independent DNA interstrand crosslink (ICL) repair

Thesis

The integrity of the genome is constantly under threat from within and without. Cells rely on the evolutionary conserved DNA damage response (DDR) mechanism to orchestrate the repair of DNA lesions and thus prevent the accumulation of mutations. One of the most deleterious types of DNA lesions, interstrand crosslink (ICL), occurs when a crosslinking agent forms covalent bonds with both strands of the double helix and tethers them together. The most well-studied among ICL repair pathways is the Fanconi Anaemia (FA) pathway, the disruption of which gives rise to a disorder of the same name. Both the FA pathway and the more recently-discovered NEIL3 pathway are replication-dependent. However, replication-independent pathways also exist and they are likely responsible for the repair of ICLs in non-dividing cells.

This thesis describes the optimisation of a purification scheme, which was designed to identify hitherto unknown players in ICL repair out of a pool of nuclear proteins. The scheme exploited the likely binding preference of such proteins for crosslinked over non-modified DNA probes. Two types of probes, linear and replication fork mimic, were designed to capture the proteins putatively associated with replication-independent and -dependent ICL repair pathways, respectively. The scheme was successfully used to identify UHRF1 and BOD1L1, two proteins with already known links to ICL repair, as well as THYN1, an EVE-domain-containing protein with no established roles in DDR. THYN1 is rapidly recruited to ICL sites in vivo and its absence confers hypersensitivity to psoralen-induced ICLs. THYN1 operates in the same pathway as FANCD2 in the repair of psoralen-derived lesions; however, unlike FANCD2, its activity is not cell cycle-dependent. Interestingly, THYN1 exerts a negative influence on MMC and cisplatin repair in FANCD2-deficient cells. Identification and analysis of putative THYN1 binding partners may help to explain this phenotype and to elucidate the molecular mechanisms which underpin THYN1-mediated repair.

Authors: Yaprianto, K

• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: DNA damage repair; DNA interstrand crosslink (ICL) repair; Fanconi Anaemia (FA) pathway; THYN1
• Subjects: DNA damage; Biochemistry