Revealing the Underlying Mechanisms of DNA Sequence Rearrangements

Using gene targeting, we have developed methods for delineating specific subpathways of homologous recombination.

We wanted to understand exactly how DNA damage leads to homologous recombination. One of our central questions was the extent to which homologous recombination results from broken replication forks versus two-ended double strand breaks. To our surprise, our experiments show that two-ended breaks are the predominant substrate for HR, despite HR's vital role in repairing broken replication forks (Jonnalagadda et al., 2005). Furthermore, we studied the mechanisms of cross-link induced homologous recombination, and found that cross-links are more likely to lead to a loss of sequence information compared to spontaneous recombination events. Intriguingly, we observed cross-link-induced single strand annealing an postulated collision of replication forks, which is now the prevailing model.

To do these mechanistic studies, we targeted our homologous recombination substrates into the ROSA locus. Currently, we are moving our ROSA recombination system from cells to mice. We have successfully targeted ES cells and created germline transgenic animals. We hope that these mice will enable analysis of tissues that have hitherto been refractory to studies of recombination.