The general goal of this project is to increase our understanding of the mechanisms that govern recombination in mammalian cells. Such an aim has broad relevance in light of the many biological roles that recombination plays. Recombination has particular relevance to the cancer problem in that aberrant genetic rearrangements and genomic instability are often associated with neoplasia.
The specific aims of this proposal focus primarily on the potential interplay between DNA mismatch repair and intrachromosomal recombination. Several aspects of how mammalian cells normally avert unwanted recombination between imperfectly matched DNA sequences will be studied. (Recombination between two sequences that are similar but not identical is referred to as """"""""homeologous recombination."""""""") We will continue to exploit our model system involving mouse Ltk (thymidine kinase deficient) fibroblasts that we have successfully used over the past several years to study a variety of parameters of intrachromosomal recombination. In our experimental system we use DNA substrates containing two defective herpes simplex virus thymidine kinase (HSV tk) genes. Each DNA construct is stably integrated into the mouse cell genome so that the two defective tk genes reside as closely linked direct repeats. Genetic selection for tk-positive segregants provides a means for detecting recombination events that reconstruct a functional HSV tk gene. Intrachromosomal recombination rates are determined by fluctuation analysis. Functional tk genes produced by recombination can also be amplified from the mouse genome using the polymerase chain reaction and subsequently analyzed at the nucleotide level. By studying genetic exchanges between HSV type 1 and HSV type 2 tk sequences, which are 81% homologous, we hope to gain insight into how homeologous recombination is regulated in mammalian cells. It is known that bacteria as well as lower eukaryotes use DNA mismatch repair activities to avoid homeologous rearrangements. Several of our experiments are geared at understanding whether DNA mismatch repair might play a similar role in mammals. Our work delves into the question of whether the formation of a predicted, highly mismatched intermediate of homeologous recombination serves as a cellular signal for a """"""""call to action"""""""" against unwanted recombination events. Our proposed experiments are particularly topical since it was recently discovered that one of the most common forms of inherited predisposition to cancer in humans is associated within defects in DNA mismatch repair. This form of cancer is also associated with instability of repeated sequences in the genome. In our current proposal we will use our experience, expertise, and knowledge gained from our previous work to study recombination in cell lines isolated from cancer patients with defects in mismatch repair. We will investigate what effects such defects may have on homologous and homeologous recombination in humans. To carry out our studies of homeologous recombination in human cancer cell lines, we have devised a novel scheme in which recombination between homeologous tk sequences is linked to the expression of the dominant, selectable neo gene. We hope to learn more about the interrelationship between DNA repair and recombination as well as increase our knowledge about the factors that may contribute to the abnormal chromosomal rearrangements seen in many different types of cancer. At the same time, we hope to gain general insight into the ways that higher eukaryotes maintain the integrity of their genomes.
| Waldman, Barbara Criscuolo; Wang, Yibin; Kilaru, Kasturi et al. (2008) Induction of intrachromosomal homologous recombination in human cells by raltitrexed, an inhibitor of thymidylate synthase. DNA Repair (Amst) 7:1624-35 |
| Waldman, A S (1994) The search for homology does not limit the rate of extrachromosomal homologous recombination in mammalian cells. Genetics 136:597-605 |
