MECHANISMS OF DNA REPAIR INDUCED MUTAGENESIS. We implemented an experimental system to determine in vivo whether DNA repair can induce mutations in flanking DNA and found that it does. In particular, the repair intermediates generated from repairing preformed normally occurring DNA mispairs on an SV40-based episome were vulnerable at a low but statistically significant frequency to an APOBEC-mediated error-prone process. SiRNA knockdowns showed that components of both the base excision repair and mismatch repair pathways, or factors that can interact with these pathways (e.g., PCNA and ATR), and TpC-preferring APOBEC cytidine deaminases, particularly A3B, are required for mutagenesis, which produces 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 be turned into 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 profiled the DNA repair enzymes and related factors in these paired cell lines and found that they differ dramatically between cells that exhibit repair-induced mutations and those that do not. We systematically evaluated the repair enzyme differences for their contribution to the mutagenic effect found that certain components the base excision repair (BER) are over expressed and likely account for the mutator phenotype in the studied breast cell line and have purified the relevant factors are examining their interaction with other repair enzymes in vitro. Comparisons between the paired cell lines also revealed several other possible novel sources of A3B substrates in addition to those generated during DNA repair. We developed in vitro systems to corroborate these suppositions and determine the biochemical mechanisms involved and now have submitted two manuscripts describing these results. We are also developing procedures to identify the proteins and factors that interact with the over expressed BER components in vivo.
| Chen, Jia; Furano, Anthony V (2015) Breaking bad: The mutagenic effect of DNA repair. DNA Repair (Amst) 32:43-51 |
| Chen, Jia; Miller, Brendan F; Furano, Anthony V (2014) Repair of naturally occurring mismatches can induce mutations in flanking DNA. Elife 3:e02001 |
| Walser, Jean-Claude; Furano, Anthony V (2010) The mutational spectrum of non-CpG DNA varies with CpG content. Genome Res 20:875-82 |
| Walser, Jean-Claude; Ponger, Loic; Furano, Anthony V (2008) CpG dinucleotides and the mutation rate of non-CpG DNA. Genome Res 18:1403-14 |
