Disintegration of Replication Forks and their Recombinational Repair

University Of Oregon Eugene, Eugene OR

Investigators

Abstract

The purpose of this research is to elucidate the mechanism of replication fork disintegration and subsequent recombinational repair in the bacterium Escherichia coli. Long-term objectives for this research are to answer the following questions: 1) What happens when a replication fork reaches a single-strand interruption, present in all DNA molecules of cells, in a template DNA? 2) What happens when a replication fork is inhibited or stalled?, and 3) How is a solitary double-stranded end repaired in E. coli? The bacterial virus lambda and E. coli chromosomes will be used to study mechanisms of these processes in vivo. Studies with the lambda chromosome will include the introduction of a single-strand nicked site and a replication termination site to see if they cause replication fork disintegration with subsequent recombinational repair. Lambda crosses with other lambda viruses will be employed to directly demonstrate DNA synthesis primed by double-strand end invasion and to study systematically the double-strand end repair reaction in vivo in terms of the reaction stages and their genetic requirements. Studies with the E. coli chromosome will include a search for recombination-dependent mutants and characterization of the replication-induced chromosomal fragmentation in conditions suspected to cause replication for disintegration. If replication fork disintegration with subsequent repair by homologous recombination is demonstrated it will explain the puzzling recombination dependence of many mutants in DNA metabolism, both in bacteria and eukaryotes. Since a disintegrated replication fork is a DNA lesion that can be repaired only by homologous recombination, its demonstration will also shed light on the inviability of recombinase-deficient cells of higher organisms.