Kinetic Analysis of Human DNA Ligase III

Abstract

DNA ligases catalyze the final step in DNA replication, repair and recombination pathways using a conserved three-step mechanism to generate a single phosphodiester bond. Humans have three LIG genes that encode the DNA ligase proteins; LIG1, LIG3 and LIG4. In the nucleus, LIG1 and LIG4 repair single- and double-strand DNA breaks, respectively. LIG3 repairs both single- and double-strand DNA breaks in mitochondria and the nucleus. In the mitochondria, LIG3 performs all the ligation events required for organellar viability. In the nucleus, LIG3 has been implicated in the error-prone alternative end-joining pathway, where it can generate a variety of chromosomal anomalies. Elucidating the biological functions of the LIG3 isozymes has been hampered by a lack of available biochemical information describing the specificity of these proteins. My thesis work describes the mechanistic framework needed for understanding the specificity of the different LIG3 isoforms, providing insight into the biological roles of the DNA ligases in DNA metabolism. Biochemical analysis of the nuclear LIG3 isozymes indicated the two enzymes were kinetically indistinguishable, suggesting their differing C-termini do not influence catalytic efficiency. When compared to LIG1, LIG3 binds Mg2+ ions 10-fold weaker, but has a 7-fold greater specificity toward the nicked DNA substrate. This increased catalytic efficiency has been attributed to the DNA binding property of the LIG3 N-terminal zinc finger domain. We determined that the zinc finger domain and a previously uncharacterized disordered linker region contribute equally to the catalytic efficiency of LIG3.¬¬¬ To elucidate the activities of the ligases for double-strand break ligation, I report the first systematic kinetic analysis of LIG1- and LIG3-catalyzed ligation of double strand breaks to elucidate their unique properties. LIG3 efficiently joined various DNA substrates under all tested conditions. In contrast, LIG1-catalyzed double-strand break ligation was highly sensitive to Mg2+ concentrations and molecular crowding. LIG1 activity rivaled that of LIG3 under optimal conditions but had a preference for 3’ overhang over 5’ overhang and blunt-ended DNA structures. Notably, LIG1 (but not LIG3) accumulated abortive ligation products under physiological conditions. Recently, the erroneous ligation activities attributed to LIG3 have implicated the enzyme as a driver of cancer progression. Moreover, LIG3 is overexpressed in cancers that require enzyme’s activity for survival, making LIG3 an attractive pharmacological target. To discover novel LIG3 inhibitors that may be useful therapeutics, I developed a fluorescence-based ligation assay that reports on DNA ligation in real-time that was validated by the kinetic characterization of LIG1- and LIG3-catalyzed ligation. I adapted this assay to a discontinuous format to conduct a high-throughput screen of small-molecule libraries. Potent and selective ligase inhibitors targeting the ligases will be useful for better understanding their roles in biology, and ligase inhibitors may one day be an effective cancer therapy.