Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Junop, Murray S.

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.

Summary for Lay Audience

Interstrand crosslinks (ICLs) are formed by chemicals that irreversibly bind both strands of a DNA helix. ICL damage prevents the separation of DNA strands, disrupting essential cellular activities, including reading genes to produce protein and copying DNA for cell division. As such, failure of a cell to quickly find and remove ICL damage can result in significant loss of genetic material, cancer or cell death. Removal of ICL damage requires the recruitment of multiple nucleases, which are molecular scissors that cleave distinct DNA structures. While different nucleases have been shown to participate in ICL repair, questions remain regarding where, when and how these nucleases function. SNM1A is a dual-function nuclease, able to cut DNA from a free end (exonuclease activity) or within a strand of unpaired DNA (endonuclease activity). Although previous reports demonstrated that SNM1A is needed for ICL repair, it remains unclear what DNA structure(s) SNM1A cuts, and with which activity. The primary objectives of this thesis were to determine how DNA structure impacts SNM1A processing and investigate the SNM1A nuclease activities required for repair. Experiments presented here demonstrate that both nuclease functions are necessary for repair. Further, identified substrate preferences of SNM1A suggest potential intermediates SNM1A may act on during repair. Finally, four molecules were found to specifically inhibit SNM1A nuclease activities in a test tube. Developing small molecules will help in future experiments to determine where and when SNM1A is acting in a cell.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

Available for download on Friday, September 01, 2023

Included in

Biochemistry Commons

Share

COinS