The major focus of our research is the relationship between mutations and human cancer. We seek to identify the sources of spontaneous mutations in normal cells and to determine whether there is an exponential increase in mutations during the growth of human cancers. Do cancer cells display a mutator phenotype and is this underlying genetic instability the basis for the progressive ability of tumors to divide where they ought not, to invade and to metastasize? Our current research efforts are in several areas, as follows.

1. Fidelity of DNA Replication. We are continuing to examine the mechanism(s) by which DNA polymerases can copy DNA with phenomenally high accuracy, approaching one error in every 10 million nucleotides polymerized. Our approach is to copy biologically active DNA in vitro and then analyze the frequency and types of mutations generated after transfection of the copied DNA into cells. An analysis of mutations produced by polymerases is key to evaluating the contribution of errors in DNA replication to spontaneous mutations. We have created mutant DNA polymerases with the following properties: high fidelity, low fidelity, ability to synthesize RNA, capacity to incorporate specific nucleotide analogs and the ability to copy DNA harboring covalently modifications. Several of these mutant DNA polymerases are being used in biotechnology.

2. A Mutator Phenoytpe in Cancer. We have hypothesized that early events in tumorigenesis involve mutations in genes that function in the maintenance of genetic stability. As a result, further mutations accumulate randomly throughout the genome of cancer cells. We have developed a series of assays to quantitate the number of randomly occurring mutations in human cancer cells.  The presence of large numbers of mutations in cancer cells can account for the ability of tumors to evade cancer chemotherapeutic agents.

3. Creation of Enzymes for Cancer Gene Therapy. We have developed methods to construct vast populations of oligonucleotides containing random sequences and to insert them into plasmids. After transfecting the plasmids into bacteria, we use genetic selection to obtain new, biologically active sequences that code for promoters and enzymes not present in nature. We have applied these methods to creation of genes encoding mutant herpes thymidine kinases that phosphorylate nucleoside analogs that terminate DNA synthesis. Our goal is to introduce the mutant genes into tumors and kill the malignant cells upon exposure of the individual to the nucleoside analog. Conversely, we have created mutant enzymes that are resistant to commonly used chemotherapeutic agents. The goal is to introduce genes encoding these enzymes into bone marrow to protect this vital organ during chemotherapy.

4. Mutations in Aging. Our research on the relationship of DNA synthesis to aging has focused on the gene that is mutated in Werner Syndrome, and on the construction of mice that express DNA polymerases with altered fidelities. Werner Syndrome is an inherited disease that is characterized by the early onset of aging and is associated with a high incidence of specific cancers. The Werner Syndrome gene encodes both DNA helicase and DNA exonuclease activities. We seek to understand how mutations in this gene result in an aging phenotype.

5. Lethal Mutagenesis of HIV. We have proposed a new strategy for anti-HIV therapy, based on increasing the mutation rate of the virus. The concept is to produce mutations by incorporating mutagenic nucleoside analogs into proviral DNA. With each cycle of viral replication, there would be a progressive increase in viral mutations, driving the mutation frequency over the threshold for viral viability. We have identified non-toxic nucleoside analogs that introduce mutations into the HIV genome and abolish HIV infection in cultured cells.  Mutagenic nucleoside analogs are now being evaluated in human clinical trials for effectiveness against HIV infection.