Chemistry and Biology of DNA Ligation

本届奖项摘要

Abstract of Current Award The overall goal of this proposal is to determine the mechanism and specificity of human DNA ligases. All organisms have an absolute requirement for DNA replication and DNA repair in order to synthesize new cells and to maintain correct cellular functions. Given its central importance, there is a great deal of interest in studying these pathways. DNA ligases are essential for DNA replication and most DNA repair pathways, however there are many fundamental questions about how DNA ligases function and our existing models lag far behind those that are available for DNA polymerases. We believe that it is essential to learn the molecular mechanism of human DNA ligases and that the insights gained from this endeavor will have significant value to human health. Some examples of where this knowledge could be useful is in patients that suffer deficiency in DNA ligase function and in abnormal states in which ligases have been overexpressed such as cancer. We will use quantitative mechanistic enzymology techniques to characterize human DNA ligase 1 (LIG1) and DNA ligase 3 (LIG3). Genetic and cellular observations suggest that these enzymes share some redundant biological functions, but that distinct functions exist for each gene product. We will extend our previous kinetic and structural studies of LIG1 to understand specificity of this enzyme and to define the minimal steps in locating and engaging a single strand break. This work will be supported by additional crystal structures that will characterize the enzyme-bound intermediates that have not been previously characterized. We have recently succeeded in producing large quantities of recombinant LIG 3 alpha and beta isoforms and we will perform a kinetic and thermodynamic characterization to understand similarities and differences with LIG1. For both enzymes, we will use site-directed mutagenesis to target specific functions, such as metal binding, DNA binding, and catalytic activity. Analysis of these mutant proteins will provide an understanding of the molecular features of eukaryotic DNA ligation that will be invaluable to understand ligase function in normal cells and in human disease