In the area of genetic recombination and DNA damage repair, we focused on the area of the accuracy of genetic recombination in general and double-strand-break repair in particular. We demonstrated that the DNA synthesis associated with genetic recombination has substantially lower fidelity than that found in general cell duplication. At least two different DNA polymerases have roles in this elevated mutation rate: Base substitutions reverting a non-sense allele are dependent on the translesion polymerase, Pol zeta, encoded by REV3, whereas reversion of frameshift alleles is REV3 independent. We investigated the mechanism of double-strand-break repair and the proteins involved in controlling the fidelity of DNA synthesis during this process. We then extended this research to demonstrate meiotic recombination is also mutagenic. DNA palindromes are thought to be a common mechanism of gene amplification found in tumor cells. However, palindromes are particularly difficult to investigate because they are difficult to clone and difficult to sequence. We created yeast strains that tolerated DNA palindromes and developed a system in which they can be generated. We also developed a method to sequence DNA palindromes. We applied those tools to the characterization of DNA palindromes isolated from human tumor cell lines.

We also studied the fidelity of transcription; again, this was historically a difficult problem. We developed a genetic screen to identify mutants with reduced fidelity. We isolated mutations that reduce the fidelity of transcription and identified mutations in several subunits of RNA polymerase that result in error prone transcription. Through collaborative studies, we studied the consequences of increased transcription error rates.

Our collaborators were Stephen Hughes, Michael Kashlev, Mark Lewandoski and Ding Jin, NCI.