Recovery of Replication Following UV-induced DNA damage

Mississippi State University, Mississippi State MS

Investigators

Abstract

Inaccurate replication in the presence of DNA damage is often responsible for cellular rearrangements and mutagenesis. Nevertheless, the cellular mechanisms by which replication-blocking lesions are processed and repaired remain largely uncharacterized. UV-induced DNA damage severely inhibits chromosomal replication. In Escherichia coli, the recovery of replication following UV irradiation is dependent on nucleotide excision repair proteins and some proteins of the recF pathway. It is speculated that several additional genes may be involved in replication recovery but they have not been examined directly in vivo. This project will determine the cellular mechanism by which chromosomal replication recovers when it is blocked by UV-induced DNA damage. The investigator will identify the structural properties of lesion-arrested replication forks throughout replication recovery. This portion of the project will be done by isolating the post-irradiation DNA from uvr mutants in sucrose gradients to quantitate the number of UV-induced lesions that can be bypassed (or skipped) by DNA replication machinery before replication arrest occurs. Post-irradiation DNA synthesis will then be analyzed using strand-specific 32P-labeled probes to determine on which template strand replication blockage occurs. In addition, the investigator will use two-dimensional agarose gel electrophoresis to characterize the structural integrity of the replication fork throughout the recovery process and identify the structural intermediates that arise at blocked replication forks during recovery. He will further identify which candidate genes are directly involved in the resumption of replication at DNA damage-blocked replication forks and characterize at which step they act. This will be done by isolating DNA synthesized during the period of replication recovery in CsCl gradients and quantifying it. The role that these candidate genes play in replication recovery will further be characterized by examining the nascent DNA degradation at blocked replication forks following UV irradiation. Through an understanding of how faithful replication resumes when it is blocked by DNA damage, the investigator hopes to identify the conditions and events which can lead to illegitimate recovery, genomic rearrangements, and lethality in the presence of DNA damage. Because changes in DNA affect all aspects of biology, these insights could profoundly affect human interactions with the natural world.