USING L1 INSERTS TO DETERMINE THE FACTORS THAT AFFECT MUTATION RATE The neutral DNA mutation rate (i.e., the accumulation of base substitutions in the absence of natural selection) is a fundamental biological parameter. Interestingly, it varies within and between chromosomes, but despite considerable study, this variation remains largely unexplained. 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 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 deaminases, particularly A3B, are required for mutagenesis which produces mutations similar to those typical of the mutator phenotypes in various cancers. Thus 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 found that reverse ChIP was not sensitive enough to isolate in sufficient quantity for analysis mismatch-containing DNA molecules that were undergoing repair. We therefore applied our episome mutation sensor to established breast cancer cell lines that contain similar A3B levels and found that some pairs differed dramatically in mutagenic repair. We are now comparing the profile of DNA repair related factors and enzymes in these cell. We are continuing our experiments to test the feasibility of deep sequencing to comprehensively examine the sequence context of the mismatch-containing DNA that affects binding of the DNA glycosylases that initiate the repair process. We also modified our Perl program we had written to detect mutational signatures in the above experimental data sets to re-examine a large dataset of orthologous chimpanzee/human L1 fossils to determine the kind of mutational processes that could underlie the neutral evolution that occurred in the primate lineage over the past 80 Myr.
| 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 |
