The role of the human endonuclease VIII-like 1 and 3 in the repair of DNA replication blocking lesions

Martin, P

Abstract

The endonuclease VIII family of DNA glycosylases initiate repair of oxidative DNA base damage through BER and have been shown to resolve DNA inter-strand crosslinks (ICLs), arising from exogenous agents. Human NEIL1 (hNEIL1) activity on psoralen generated mono-adducts (MAs) and three-stranded DNA ICLs was previously described and recently NEIL3 from Xenopus laevis was reported to repair psoralen generated MAs and apurinic/apyrimidinic site DNA ICLs. Here, complementary roles of hNEIL1 and hNEIL3 in the removal of two oxidized pyrimidine bases were identified within a model DNA replication fork structure in vitro. Further investigation revealed a novel activity of truncated and full-length recombinant hNEIL3 proteins in vitro on single-stranded psoralen MAs and three-stranded and four- stranded psoralen ICL DNA substrates, highly toxic replication stalling lesions. For the first time it was shown that NEIL1/3 possess activity on four-stranded DNA ICLs. With the hNEIL3 catalytic knockout mutation K81A, elimination of activity on three- and four-stranded DNA ICLs was confirmed. hNEIL1 was recently shown to be ubiquitinated by the E3 ubiquitin-ligase, tripartite motif-containing protein 26 (TRIM26). Here, TRIM26 was identified as the major ubiquitin ligase for hNEIL3 through HeLa cell fractionation and in vitro ubiquitination assays. TRIM26 RNAi in U2OS cancer cells increased steady state levels of hNEIL1/3 and resulted in resistance to the ICL inducing agent cisplatin, determined through clonogenic survival assays. Transient overexpression of hNEIL1/3 complemented the observed phenotype. Finally, a model is proposed for incision-independent ICL repair in mammalian cells. FANCM - facilitated replication traverse of an ICL is envisaged, followed by NEIL1/NEIL3 initiated repair of ICL structures, independent of the induction of double-strand DNA breaks. This pathway is proposed to be modulated by TRIM26, providing resistance to ICL-inducing agents in cancer cells which display ubiquitin proteasome dysfunction.

Citation

Martin, P. (in press). The role of the human endonuclease VIII-like 1 and 3 in the repair of DNA replication blocking lesions. (Thesis). University of Salford

• Thesis Type: Thesis
• Acceptance Date: Dec 14, 2018
• Deposit Date: Dec 18, 2018
• Publicly Available Date: Dec 18, 2018
• Additional Information: Projects : The Pathway to Excellence Studentship