The long range goal of the proposal is to elucidate the molecular mechanisms whereby the proteins involved in DNA replication interact with nucleic acids and precursors of nucleic acids. The specific objective is to study these interactions in DNA polymerase I of Escherichia coli using a newly developed laser cross-linkin methodology. The laser cross-linking technique allows molecular contacts between proteins and DNA to be "frozen" by the formation of a covalent bond. Bond formation is induced by a rapid (8 nanosecond) pulse from the ultraviolet region of the spectrum. The brevity of bond induction effectively "freezes" the equilibrium between proteins and nucleic acids. The laser cross-linking technique will be used to define contacts and affinities between the various substrate binding sites of DNA polymerase I and the substrates themselves. Defining these contacts and affinities in parallel with studies of kinetic parameters (processivity, synthesis rate, exonuclease rates, etc.) should greatly aid our understanding of how the polymerase works in its various modes (working "forward" as a polymerase and 5' to 3' exonuclease or working "backward" as a 3' to 5' exonuclease) and how the "forward" and "backward" reactions are regulated with respect to each other. Additionally, there exist a number of mutations in DNA polymerase I that alter its function. These mutant proteins will be examined to determine how the altered function is related to structural interactions. After defining how multiple catalytic activities are coordinated in a single polypeptide, the studies will be extended to more complex systems where more than one protein is required for assembly and/or function of a DNA replication complex. An understanding of DNA replication may give us an indication of where normal systems go awry in cancer and various genetic disorders. The information gleaned from defining specific contacts may be useful in designing drugs that prevent or control certain disease conditions.
