Analysis of Single DNA Polymerase Complexes at 5 Angstrom Precision in Real Time

点击翻译按钮获取中文摘要

DESCRIPTION (provided by applicant): Project Summary This application aims to achieve electronic control of DNA polymerase function on a time scale that superimposes with rates of enzyme binding and catalysis. To achieve this aim, we will monitor the interaction of individual DNA polymerases with a nanopore sensor under voltage- induced tension. We will characterize kinetic, biochemical, and structural properties of polymerase-DNA complexes captured under voltage control in a nanopore. We will optimize nanopore measurements of polymerase function at significantly higher bandwidth than is possible using conventional techniques and in a manner that permits serial analysis of thousands of individual enzymes as they process DNA. We believe the study is innovative because it will employ a recently established nanopore technique to identify and measure translocation steps during individual catalytic cycles of replication. Discrimination between polymerase-driven translocation mechanisms should be achievable. This work is relevant to human health because mutations that arise from misincorporation of nucleotides by DNA polymerases are a fundamental cause of cancer. In addition, nanopore-coupled polymerases could present a high speed, low cost technology for genome sequencing that has virtually no environmental impact. PUBLIC HEALTH RELEVANCE: This work focuses on mechanisms of DNA replication by DNA polymerases. It is relevant to human health because mutations that arise from misincorporation of nucleotides by DNA polymerases are a fundamental cause of cancer. In addition, nanopore-coupled polymerases could present a high speed, low cost technology for genome sequencing that has virtually no environmental impact.