Elucidating Restriction Endonucleases Reaction Mechanisms via Dwell-Time Distribution Analysis

Abstract

We have developed a Total Internal Reflection Fluorescence (TIRF) microscopy based assay that allows us to simultaneously measure the length of the catalytic cycle for hundreds of restriction endonuclease molecules in one experiment. We stably attach thousands of short duplex DNA molecules, each labeled with a single quantum dot semiconductor nanocrystal, to a passivated glass surface within a flow channel. The disappearance of a quantum dot indicates that its DNA tether has been cleaved. We introduce restriction endonuclease molecules into the channel in the absence of magnesium, which permits binding to, but not cleavage of the surface immobilized DNA substrate. When buffer containing magnesium is introduced into the flow channel, DNA cleavage by the pre-bound restriction endonuclease molecules is initiated. This synchronization allows us to measure the lag time between the introduction of magnesium and the completion of DNA cleavage for the entire population of enzymes. Analysis of the dwell-time distributions can provide insights into the DNA cleavage mechanism. Our observations suggest that EcoRV, a dimeric Type II restriction endonuclease that cleaves the palindromic sequence GAT↓ATC (where ↓ is the cut site), requires two kinetic steps to complete duplex cleavage after prebinding. However, dwell-time distributions suggest that BcnI, which is active as a monomer and cleaves the pseudopalindromic sequence 5'-CC↓SGG-5' (where S stands for C or G), requires more than four kinetic steps to complete duplex cleavage. Furthermore, experiments performed with strand-specific DNA substrates suggest that the number of steps indicated by the dwell-time distribution depends on which strand of the recognition site must be cleaved to result in quantum dot release. By designing additional substrates that mimic the various intermediate states, we plan to dissect the mechanism by which BcnI cleaves each strand of the intact restriction site.