The Role of Divalent Metal Ions in Enzymatic DNA Ligation

DNA Ligase I;DNA Repair;Enzyme Kinetics;Biological Chemistry;Health Sciences;Biological Chemistry

Taylor, Mark RobertPalfey, Bruce A. ;

University of Michigan

关键词: DNA Ligase I;    DNA Repair;    Enzyme Kinetics;    Biological Chemistry;    Health Sciences;    Biological Chemistry;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/108846/trotsky_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Human DNA ligase I (LIG1) catalyzes the formation of a phosphodiester bond between adjacent DNA strands during nuclear DNA replication and single-strand break repair.The kinetic mechanism of enzymatic DNA ligation involves three independent phosphoryl transfer steps, each of which requires the presence of divalent metal ions. This thesis reports the first in-depth characterization of the basic kinetic mechanism of enzymatic DNA ligation in the presence of magnesium, the presumptive physiological cofactor. Thischaracterization serves as a basic framework against which future studies of the enzyme can be compared. This work also gives the first description of the occurrence of abortive DNA ligation on clean DNA breaks. These abortive ligation intermediates appear to be a causative agent in the neurodegenerative disease Ataxia with Oculomotor Apraxia 1. We explore the interaction of divalent metals with LIG1 by comparing the kinetics of enzymatic ligation in the presence of the alternative metal manganese to what was observed with magnesium. Manganese fulfills all catalytically required roles, however it also inhibits the enzyme at elevated metal concentrations. Inhibition is not competitive, suggesting the presence of an inhibitory metal binding site outside of the active-site. We further investigate the function of metal ions during catalysis by LIG1 by characterizing the effect of mutations at putative metal binding sites within the enzyme active site. We find that mutations to residues coordinating metal sites on opposite sides of the nick produce nearly identical effects on adenylyl transfer and nick-sealing. This supports the model that there is a rearrangement of the DNA intermediates between the adenylyl transfer and nick sealing steps to allow a common core of two divalent metal ions to catalyze both DNA-dependent steps.