Dual Functions of Interstrand Crosslink Repair Nuclease SNM1A
Abstract
Interstrand crosslinks (ICL) are a highly cytotoxic form of DNA damage, covalently linking opposing strands of DNA. ICLs disrupt essential cellular processes requiring strand separation, including transcription and replication. Consequently, lesion recognition and removal are critical to prevent chromosomal aberrations, mitotic catastrophe and apoptosis. ICL repair requires the coordination of a complex network of nucleases necessary for remodelling, unhooking and resolving repair intermediates. While many nucleases participate, little is known about where and when each nuclease acts. SNM1A is a dual-function exonuclease and endonuclease necessary for ICL repair. Where SNM1A is absent, cells accumulate irreparable double-strand breaks and exhibit reduced survival following treatment with ICL-inducing compounds. Although essential for fidelitous repair of ICLs, it is unclear where SNM1A functions and which intermediate(s) it processes.
The primary objectives of this thesis were to examine the capacity and preferences of SNM1A nuclease activities in vitro, investigate which nuclease activities contribute to ICL repair and develop small molecule inhibitors of SNM1A. To examine functional preferences, we characterized SNM1A nuclease activities on various potential repair intermediates. While SNM1A exonuclease activity was generally more robust than the endonuclease function, translesional processing constituted the rate-limiting step during digestion of an ICL-containing stalled replication fork mimic. Further, structural models of SNM1A and its yeast homolog were generated to enable mutagenic isolation of nuclease functions. SNM1A exonuclease and endonuclease processing were selectively disrupted by substituting residues in the phosphate-binding pocket and novel DNA binding groove, respectively. In a yeast model, neither separation-of-function mutant was sufficient to facilitate ICL repair, indicating that both nuclease activities are necessary. Finally, an in silico high-throughput screen identified four specific inhibitors of SNM1A with low micromolar potency. Cumulatively, experiments presented in this thesis expand the potential roles of SNM1A in ICL repair and provide promising lead compounds to target SNM1A in vivo.