Defining the role of endonuclease VIII-like 1 and 3 in the repair of interstrand crosslinks in cancer cells

Burberry, G

Abstract

Fanconi Anaemia (FA) is an inherited autosomal-recessive disorder that can lead to abnormal development, bone-marrow failure, and an increased vulnerability to carcinogenesis. Cells derived from FA patients are unusually sensitive to DNA crosslinking agents and it is now known that FA cells lack one of twenty-two known different FA complementation group (FANC) proteins involved in the repair of DNA interstrand crosslinks (ICLs). More recently, two DNA glycosylases (endonuclease VIII – like 1 & 3 [NEIL1 & NEIL3]) that excise oxidised bases from DNA, thus initiating base excision repair, have been found to participate in the resolution of psoralen induced ICLs. Therefore, this project set out to determine whether the modulation of NEIL1 or NEIL3 expression in cell lines lacking FANCD2, of the monoubiquitinated DNA-heterodimer protein complex FANCD2/FANCI, could significantly affect their sensitivity to ICL-inducing agents, such as mitomycin C and cisplatin. An FA original and two FA-generated cancerous cell model systems representing FA and equivalent wildtype cells were then transfected with plasmids expressing FLAG-tagged NEIL1 or NEIL3 and challenged with the crosslinking agents or the oxidising agent tert-butyl hydroperoxide, and any differences in sensitivity to these agents determined. Similarly, siRNAs were designed against NEIL3 and the E3-ubiquitin ligase tripartite motif-containing protein 26 (TRIM26), that has been shown to ubiquinate both NEIL1 and NEIL3 for proteasomal degradation and similar cell growth analyses performed using the MTT assay. Recombinant NEIL1 expression was confirmed by western blotting but had little effect on resistance to crosslinking and oxidising agents. Overexpression of NEIL3, either a truncated version lacking the C-terminal GRF domains (NEIL31506), or the full-length protein, proved more difficult to confirm by western blotting, possibly due to antibody specificities to recombinant and non-recombinant NEIL3. However, MTT assays generally indicated a small increase in resistance irrespective of FA background, suggesting that NEIL3 overexpression was achieved in these cells. Unexpectedly, compared to literature and Elder laboratory results, knockdown of NEIL3 showed increased resistance against ICL and oxidative agents independent of FA phenotype, while knockdown of TRIM26 showed no clear effect on the sensitivity of the FA cell line to the genotoxic agents tested. In conclusion, the results indicate that the overexpression of NEIL1 or NEIL3 could not compensate for the loss of the FA pathway of ICL repair and had little reproducible effect on the resistance of these cell lines to crosslinking and oxidizing agents.

Citation

Burberry, G. Defining the role of endonuclease VIII-like 1 and 3 in the repair of interstrand crosslinks in cancer cells. (Thesis). University of Salford

• Thesis Type: Thesis
• Deposit Date: Aug 5, 2022
• Publicly Available Date: Aug 5, 2022
• Award Date: Jul 1, 2022