AbstractMammalian SLX4 is a scaffold protein that directs the formation of a multi-protein DNA processing toolkit that includes three different structure-selective endonucleases: XPF-ERCC1, MUS81-EME1 and SLX1. The SLX4 complex has been shown to have essential roles in repair of DNA interstrand crosslinks (ICLs). In addition to its role in ICL repair, the SLX4 complex has the relatively unusual property of being able to cleave efficiently four-way DNA structures called Holliday junctions (HJs) in vitro. At the outset of this study a major unanswered question was how the binding to SLX4 influences the associated nucleases in functional terms.
In the first chapter, I focus on the regulation of MUS81-EME1 by the SLX4 scaffold in ICL repair. I describe the identification of point mutations in MUS81 that abolish the interaction with SLX4. Complementation studies in Mus81 null MEFs suggested that the role of MUS81-EME1 in ICL repair is independent of its interaction with SLX4. In contrast, the interaction of MUS81-EME1 with SLX4 is essential for ICL repair in two different human cell lines. Finally, I show that in human cells, generation of ICL-induced DSBs does not require MUS81-EME1. However, the repair of these DSBs is dependent on MUS81-EME1 and requires binding of MUS81 to the SLX4 scaffold.
In the last two chapters, I investigate the role of the SLX4 scaffold in ICL repair and HJ resolution. To this end, I identify separation-of-function SLX4 mutants that abolish interaction with MUS81-EME1, SLX1, XPF-ERCC1 or a combination of these nucleases. Complementation studies in Slx4 null MEFs revealed that the role of SLX4 in ICL repair is primarily dependent on its interaction with XPF-ERCC1 and partly on its regulation of the SLX1 nuclease. Consistent with my observations in Mus81 null MEFs, SLX4 interaction with MUS81-EME1 seems to be dispensable for ICL repair in mice. However, the tethering of MUS81-EME1 and SLX1 nucleases to the SLX4 scaffold is essential for HJ resolution in the absence of the BLM dissolution pathway. Thus, the SLX4 scaffold plays a pleiotropic role in the maintenance of genome integrity by targeting specific nucleases to sites of ICL repair and HJ resolution.
|Date of Award||2014|
|Supervisor||John Rouse (Supervisor)|