Veronica M. Maher, Ph.D.

maher@msu.edu

Research

My research, which is closely related to that of Dr. J. Justin McCormick, concentrates on determining the molecular and cellular mechanisms involved in the induction of the DNA changes (mutations) that occur in human cells when they are exposed to ultraviolet or ionizing radiation or chemical carcinogens. In particular, my group examines the role of DNA replication, genetic mitotic homologous recombination, and various kinds of DNA repair mechanisms. For example, we obtain cells that are deficient in one or other of these processes, i.e., DNA replication, genetic recombination, nucleotide excision repair, mismatch repair, p53, etc., or use genetic techniques to engineer cells that lack gene products involved in these processes. We compare these cells with normal human cells for the frequency and spectrum of mutations induced in the target HPRT gene by various carcinogens. The target cell strains are treated with the carcinogen and selected for those with mutations. We use RT-PCR to amplify HPRT cDNA directly from the mutant clone, sequence the DNA, determine the nature of the mutation and its location in the gene (spectrum), and deduce the premutagenic lesion responsible for the mutation. To determine if the sites of the mutations correspond to the sites of the initial DNA damage or damage remaining unrepaired, various techniques are employed. These include using E. coli UvrABC excinuclease or T4 endonuclease to cut the gene at the site of carcinogen adducts or photoproducts, followed by ligation-mediated PCR to map the location of the damage at the nucleotide level.

As an alternate system, cell free extracts are prepared from normal cells and from cells defective in one or other of the processes referred to above are used to replicate carcinogen-damaged DNA in vitro. The products of replication are analyzed for the frequency and kinds of mutations induced by specific types of damage at specific sites in the DNA. Using both systems we have been studying the role of the human polymerase zeta in human cell mutagenesis and also the mechanisms responsible for the abnormally high frequency and highly unusual spectrum of mutations observed with cells from xeroderma pigmentosum variant patients.

To study the mechanisms of carcinogen-induced genetic recombination, we use human cells engineered to contain two defective copies of a selectable gene, stably integrated into a chromosome. Only cells that successfully combine the first part of one gene and the last part of the other gene can survive selection and form a colony. The system gives us a quantitative measure of the frequency of recombination induced by various carcinogens, and allows us to determine the nature of the recombination event. Integration of this recombination substrate into human cells that are deficient in various processes such as nucleotide excision repair, mismatch repair, p53, etc., or lack a particular DNA polymerase enables us to determine the role of these processes in recombination.

The research is carried out by a team of ~20 persons, including professors, postdoctoral research associates, and graduate students from the basic science areas such as microbiology biochemistry, cell and molecular biology, genetics, and pharmacology and toxicology. They are assisted by research assistants and undergraduate students. Funding comes mainly from federal grants for research. Students interested in carcinogenesis and these related basic science areas are encouraged to discuss their interests with one of the directors and carry out a rotation in the laboratory to become acquainted with the research being conducted.