Journal
ACS NANO
Volume 3, Issue 4, Pages 995-1003Publisher
AMER CHEMICAL SOC
DOI: 10.1021/nn9000897
Keywords
nanopore; alpha-hemolysin; Klenow fragment; DNA-binding proteins; single molecule; active control
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Funding
- National Institutes of Health [GM23950-444071, HG004035-03]
- NATIONAL HUMAN GENOME RESEARCH INSTITUTE [K25HG004035] Funding Source: NIH RePORTER
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DNA polymerases catalyze template-dependent genome replication. The assembly of a high affinity ternary complex between these enzymes, the double strand-single strand junction of their DNA substrate, and the deoxynucleoside triphosphate (dNTP) complementary to the first template base in the polymerase active site is essential to this process. We present a single molecule method for iterative measurements of DNA-polymerase complex assembly with high temporal resolution, using active voltage control of individual DNA substrate molecules tethered noncovalently in an alpha-hemolysin nanopore. DNA binding states of the Klenow fragment of Escherichia coli DNA polymerase I (KF) were diagnosed based upon their ionic current signature, and reacted to with submillisecond precision to execute voltage changes that controlled exposure of the DNA substrate to KF and dNTP. Precise control of exposure times allowed measurements of DNA-KF complex assembly on a time scale that superimposed with the rate of KF binding. Hundreds of measurements were made with a single tethered DNA molecule within seconds, and dozens of molecules can be tethered within a single experiment. This approach allows statistically robust analysis of the assembly of complexes between DNA and RNA processing enzymes and their substrates at the single molecule level.
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