Title: Structure and mechanism of human DNA ligase III, an essential repair enzyme
Description: DNA ligase III is one of three human DNA ligases responsible for repairing breaks in the backbone of DNA, which if left unrepaired can lead to genomic instability, cancer and symptoms related to accelerated aging. Understanding the recruitment of DNA ligase III to DNA is essential to understanding the regulation of DNA repair in vivo. DNA ligase III contains a unique N-terminal zinc finger domain that binds to nicks and gaps in DNA. This small domain has been described as a nick sensor, but it is not required for DNA nick joining activity in vitro. Recent structural studies of other human DNA ligases identified other domains in DNA ligase III that potentially participate in DNA binding and nick recognition.

This dissertation describes the DNA binding and ligation activities of human DNA ligase III. The DNA binding affinity and specificity of each domain of DNA ligase III were measured using biochemical methods. These studies identified two separate, independent DNA binding modules in DNA ligase III that each bind specifically to nicked DNA over intact duplex DNA. Both binding modules are required for ligation of blunt ended DNA substrates. Although the zinc finger increases the catalytic efficiency of nick ligation, it appears to occupy the same binding site as the DNA ligase III catalytic core. Crystallization of a zinc finger deletion mutant showed that DNA ligase III fully encircles the DNA and sequesters the nick in the enzyme active site, presumably to the exclusion of the zinc finger. This suggests that the zinc finger functions at an earlier step of end joining, initially serving to recruit DNA ligase III to the site of a nick in DNA then disengaging to allow access by the other domains during the subsequent reactions (DNA adenylation and nick ligation) that join the ends of the DNA together. These structural and mechanistic studies explore the molecular properties of DNA Ligase III involved in its recruitment to specific sites of DNA damage, and offer insight into the coordination of DNA repair transactions in vivo.