Mammalian L1 retrotransposons as genetic characters

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

MECHANISMS OF DNA REPAIR INDUCED MUTAGENESIS. We had implemented an experimental system to determine in vivo whether DNA repair can induce mutations in DNA that flanked the site of DNA repair and found that it does. In particular, the repair intermediates generated from repairing preformed normally occurring DNA mispairs that we introduced into an SV40-based episome were vulnerable at a low but statistically significant frequency to an APOBEC3 (A3) C-deaminase-mediated error-prone process. Both base excision repair (BER) and mismatch repair pathways (MMR) were involved in generating the obligatory single stranded substrate for TpC-preferring A3B deaminase. This process produced mutations similar to those typical of the mutator phenotypes in various cancers. The mechanistic basis of the mutator phenotype is not known but our studies suggested that normally error-free DNA repair processes can act as mutators providing a heretofore unexpected source of genetic changes that underlie disease, aging and evolutionary change. We applied our episome mutation sensor to pairs of established breast cancer cell lines that contain similar A3B levels and found that some pairs differed dramatically in mutagenic repair. We found that this difference was due to elevated expression of the bifunctional DNA glycosylase, NEIL2, which sensitizes breast cancer cells to both A3B-mediated mutations and double strand breaks (DSBs) by perturbing canonical BER. NEIL2 is normally involved in the removal of oxidized bases, which can accumulate under conditions of oxidative stress that often occurs in cancer cells. We showed that NEIL2 usurps the canonical BER DNA lyase, APE1, at abasic sites in a purified BER system, and that the nicked NEIL2 product is a poor substrate for the next enzyme in the BER pathway, beta polymerase. However, the nicked NEIL2 product can serve as an entry site for the MMR enzyme Exo1 in vitro to generate single-stranded DNA, which would be susceptible to both A3B and DSBs. Our findings that NEIL2 or Exo1 depletion mitigates the DNA damage caused by A3B expression, indicates that aberrant NEIL2 expression can abort normal BER and drive its intermediates into an MMR pathway to generate A3 substrates. In vivo evidence for the involvement of MMR in A3-mediated mutations has reccently been published (Mas-Ponte & Supek: DNA mismatch repair promotes APOBEC3-mediated diffuse hypermutation in human cancers. Nature Genet. 2020). Preliminary experiments using mass spectrometry to profile NEIL2-interacting proteins under conditions of oxygen stress and RNA-seq to examine the effect of oxygen stress on the transcriptome were inconclusive.