March 12th, at 3:00pm, LS302
Chemicals and Cancer: Mechanism of Replicating Carcinogen-Damaged DNA
Presented by Wayne State University Professor of Biochemistry, Dr. Louis J. Romono
Abstract
One of the major goals of this work is to gain an understanding of the molecular mechanism of DNA replication and how DNA polymerases deal with carcinogenic adducts on DNA. Studying the relationship between adduct structure within a specific sequence context and the biochemical effects that are induced should help to clarify the molecular mechanism of mutagenesis and may lead to an understanding of why certain adducts can be accommodated into the polymerase active site allowing bypass synthesis while others block synthesis. During DNA replication, DNA polymerases incorporate a nucleotide through a multi-step mechanism that involves a conformational change from an open binary to a closed ternary complex. This closed complex is able to catalyze the requisite chemical step to form the phosphodiester bond. We have been able to detect the formation of a closed complex of DNA polymerase I bound to a primer-template in presence of the next correct dNTP using a limited proteolysis method. This conformational change, which is believed to contribute to the high fidelity of DNA replication, is affected by the presence of a DNA adduct or by a mispaired dNTP. In addition, we have used a gel-retardation assay to show that the ternary complex is more stable than the open one and that this stability is affected by a DNA adduct located within the polymerase active site. Finally, we have prepared polymerase mutants and have shown that specific amino acid residues within the active site alter both the polymerase conformational change and binding strength.
Seminar Calendars
Fall 2003
Spring 2004
