Mammalian L1 retrotransposons as genetic characters

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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. 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 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 are now systematically evaluating the repair enzyme differences for their contribution to the mutagenic effect or lack thereof and will supplement these studies with WGA of the paired cell lines to determine if they harbor mutational signatures consistent with those obtained with our episomal reporter. 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 have developed in vitro systems to corroborate these suppositions and determine the biochemical mechanisms involved. We are also modifying our Perl program that we had written to detect mutational signatures in the above episomal based data sets to re-examine a large dataset of orthologous chimpanzee/human L1 fossils to determine the kinds of mutational processes that could underlie the neutral evolution that occurred in the primate lineage over the past 80 Myr as a function of CG content.