Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein

Summary

Msh2-Msh6 is responsible for initiating repair of replication errors in DNA. Here we present a transient kinetics approach towards understanding how this critical protein works. The report illustrates stopped-flow experiments for measuring the coupled DNA binding and ATPase kinetics underlying Msh2-Msh6 mechanism of action in DNA repair.

Abstract

Transient kinetic analysis is indispensable for understanding the workings of biological macromolecules, since this approach yields mechanistic information including active site concentrations and intrinsic rate constants that govern macromolecular function. In case of enzymes, for example, transient or pre-steady state measurements identify and characterize individual events in the reaction pathway, whereas steady state measurements only yield overall catalytic efficiency and specificity. Individual events such as protein-protein or protein-ligand interactions and rate-limiting conformational changes often occur in the millisecond timescale, and can be measured directly by stopped-flow and chemical-quench flow methods. Given an optical signal such as fluorescence, stopped-flow serves as a powerful and accessible tool for monitoring reaction progress from substrate binding to product release and catalytic turnover^1,2.

Here, we report application of stopped-flow kinetics to probe the mechanism of action of Msh2-Msh6, a eukaryotic DNA repair protein that recognizes base-pair mismatches and insertion/deletion loops in DNA and signals mismatch repair (MMR)^3-5. In doing so, Msh2-Msh6 increases the accuracy of DNA replication by three orders of magnitude (error frequency decreases from ~10^-6 to10^-9 bases), and thus helps preserve genomic integrity. Not surprisingly, defective human Msh2-Msh6 function is associated with hereditary non-polyposis colon cancer and other sporadic cancers^6-8. In order to understand the mechanism of action of this critical DNA metabolic protein, we are probing the dynamics of Msh2-Msh6 interaction with mismatched DNA as well as the ATPase activity that fuels its actions in MMR. DNA binding is measured by rapidly mixing Msh2-Msh6 with DNA containing a 2-aminopurine (2-Ap) fluorophore adjacent to a G:T mismatch and monitoring the resulting increase in 2-aminopurine fluorescence in real time. DNA dissociation is measured by mixing pre-formed Msh2-Msh6 G:T(2-Ap) mismatch complex with unlabeled trap DNA and monitoring decrease in fluorescence over time⁹. Pre-steady state ATPase kinetics are measured by the change in fluorescence of 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl) coumarin)-labeled Phosphate Binding Protein (MDCC-PBP) on binding phosphate (Pi) released by Msh2-Msh6 following ATP hydrolysis^9,10.

The data reveal rapid binding of Msh2-Msh6 to a G:T mismatch and formation of a long-lived Msh2-Msh6 G:T complex, which in turn results in suppression of ATP hydrolysis and stabilization of the protein in an ATP-bound form. The reaction kinetics provide clear support for the hypothesis that ATP-bound Msh2-Msh6 signals DNA repair on binding a mismatched base pair in the double helix.

F. Noah Biro and Jie Zhai contributed to this paper equally.

Protocol

A. Measurement of Msh2-Msh6 DNA Binding Kinetics

1. Sample preparation for the Msh2-Msh6 DNA binding kinetics experiment

Preparation of reagents for a fluorescence-based kinetic DNA binding experiment on a stopped-flow is similar to that for an equilibrium experiment on a fluorometer. Indeed equilibrium binding analysis should be performed first to estimate the dissociation constant (K_D) for the interaction in order to optimize reaction conditions for kinetic analysis. Stopped-flow experiments require larger quantities of biological materials compared with equilibrium or steady-state experiments;....

Discussion

The example of a DNA mismatch binding protein described here illustrates the power and utility of transient kinetic methods for studying the mechanisms of biological molecules. Stopped-flow measurements on the single turnover time scale provided unambiguous evidence for rapid and specific binding of Msh2-Msh6 protein to a mismatched base pair and formation of a long-lived protein X DNA complex in the reaction ⁹. Moreover, stopped-flow (and quench-flow) analysis of ATPase activity provided direct evidence for M.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
37 G			DNA Sequence: 5’- ATT TCC TTC AGC AGA TAT G T A CCA TAC TGA TTC ACA T -3’
37 T (2-Ap)			DNA Sequence: 5’- ATG TGA ATC AGT ATG GTA TApT ATC TGC TGA AGG AAA T -3’
37 T			DNA Sequence: 5’- ATG TGA ATC AGT ATG GTA T A T ATC TGC TGA AGG AAA T -3’