Budding yeast Slx4 binds to the structure-specific DNA repair nucleases Slx1 and Rad1XPF-Rad10ERCC1, and it was reported that Slx4 is essential for DNA flap cleavage by Rad1XPF-Rad10ERCC1 during certain types of DNA repair in yeast. At the outset of this thesis, bioinformatic analyses identified the uncharacterised protein BTBD12 in higher eukaryotes as a putative orthologue of yeast Slx4. In the first results chapter of this thesis, I describe the identification of BTBD12-interacting proteins, including XPF-ERCC1 and SLX1. These findings led me to refer to BTBD12 as human SLX4. I found that SLX4 binds to another structure-specific nuclease MUS81-EME1, and other proteins involved in telomere maintenance and cell cycle progression. The remainder of this chapter describes detailed biochemical analysis of the nuclease activities associated with the SLX4 complex isolated from human cells. Work from this lab and others revealed that depletion of SLX4 from human cells using siRNAs causes defects in the repair of DNA interstrand crosslinks (ICLs). Inherited mutations in humans that reduce the efficiency of ICL repair cause Fanconi anaemia (FA). The cellular sensitivity of SLX4 depleted cells to ICLs prompted me to investigate SLX4 as a candidate FA gene. Dr. Johan de Winter (VU University Medical Center, Amsterdam) and Dr. Detlev Schindler (University of Wurzburg) had identified several patients with unclassified FA that was not caused by mutations in the FA genes known at the time. In the second results I describe characterisation of SLX4, and the SLX4 holo-complex, in cells from some of these FA patients who had bi-allelic SLX4 mutations. In three of the patients SLX4 was expressed at normal levels but was missing part of the first, and all of the second, UBZ-type putative ubiquitin-binding domain. This prompted me to investigate the function of the SLX4 UBZ domains. I found that the first, but not the second, UBZ domain of SLX4 binds to ubiquitin in vitro and targets SLX4 to sites of DNA damage in vivo. Furthermore, the first but not the second SLX4 UBZ domain appears to be required for ICL repair, demonstrating the important of correctly localising SLX4 for DNA repair. In the final chapter, I present preliminary data which suggests that SLX4 is regulated in an unusual manner in during S-phase of the cell cycle, and that SLX4 interacts with the PLK1 kinase in a phosphorylation-dependent manner.

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<p>Interstrand cross-linking (ICL) damage to DNA is cytotoxic as it blocks replication and transcription. This cytotoxicity is exploited in anti-cancer therapies, but increased ICL repair limits the efficacy of these chemotherapies. SNM1A (sensitive to nitrogen mustard 1A), of the beta-CASP family of nucleases, has been shown to participate in the initiation of one of the ICL repair processes. Biochemical studies of SNM1A have been limited due to insolubility and instability of SNM1A in bacteria and insect cell lines and toxicity in human cell lines. Work reported in this thesis describes a novel and efficient method of generating active protein from inclusion body expression of the beta-CASP domain of SNM1A. This refolded beta-CASP domain shows 5’ exonuclease activity on single stranded and double stranded DNA in vitro. Nevertheless, this domain alone is unable to complement <em>pso2</em> null ICL repair defects in<em> S. cerevisiae</em> after exposure to ICL agents. These functional studies of the beta-CASP domain of SNM1A will be helpful in directing future research on its role in ICL repair. Additionally, this will aid future structural and inhibitor studies of this essential interstrand cross-link repair protein, SNM1A.</p> / Master of Science (MSc)

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