Home Research Labs Lohman Lab Lohman Lab Kinetics and mechanism of polynucleotide ligases

Kinetics and mechanism of polynucleotide ligases

DNA ligases catalyze the formation of a phosphodiester bond between an adjacent 5’ PO4 and 3’ OH in three distinct catalytic steps: a) self-adenylation b) adenylyl transfer and c) phosphodiester bond formation. Weuse rapid chemical quench, stopped-flow, and bulk fluorescence measurements to determine the microscopic kinetic rates that drive each of these catalytic events in several viral, archaeal and human DNA ligases. Our work seeks to understand the mechanistic details of DNA binding, ligation site localization, conformational changes during turnover, and the chemical steps of ligation. Further, we aim to understand not only the biologically relevant nick-sealing event, but also end-joining in the absence of accessory factors, an activity critical for biotechnological ligation applications.

 

Bauer, R.J., et al (2017). ACS Pubs. doi.org/10.1021/acs.biochem.6b01261

 

We have several ongoing collaborations aimed at broadening our approach to these questions. In collaboration with the Hoskin’s lab at University of Wisconsin-Madison, we are using fluorescently labeled DNA ligases, single-molecule FRET and bulk FRET, to investigate the real-time conformational changes of DNA ligases during the process of ligation and to probe ligase-nucleic acid binding interactions. With the Keck and Grant labs, also at University of Wisconsin-Madison, we aim to capture DNA ligase structures bound to DNA end-joining and other substrates.

We additionally use highly multiplexed single molecule sequencing assays to profile the substrate specificity of DNA ligases on end-joining substrates. Our single molecule sequencing method allows us to investigate the ability of DNA ligase to discriminate against mismatches (fidelity) and sequence preferences of the enzyme (bias). We are extending this analysis to profile a variety of DNA ligases and end types, as well as to develop a deeper understanding of kinetics and thermodynamics of ligation.

 

graphs showing normalized read frequencies, mismatches, Watson-Crick matches and overhangs

Duckworth, A.T., et al (2023), Current Protocols. doi.org/10.1002/cpz1.690