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Replication of individual DNA molecules under electronic control using a protein nanopore.

ABSTRACT: Nanopores can be used to analyse DNA by monitoring ion currents as individual strands are captured and driven through the pore in single file by an applied voltage. Here, we show that serial replication of individual DNA templates can be achieved by DNA polymerases held at the ?-haemolysin nanopore orifice. Replication is blocked in the bulk phase, and is initiated only after the DNA is captured by the nanopore. We used this method, in concert with active voltage control, to observe DNA replication catalysed by bacteriophage T7 DNA polymerase (T7DNAP) and by the Klenow fragment of DNA polymerase I (KF). T7DNAP advanced on a DNA template against an 80-mV load applied across the nanopore, and single nucleotide additions were measured on the millisecond timescale for hundreds of individual DNA molecules in series. Replication by KF was not observed when this enzyme was held on top of the nanopore orifice at an applied potential of 80 mV. Sequential nucleotide additions by KF were observed upon applying controlled voltage reversals.

SUBMITTER: Olasagasti F

PROVIDER: S-EPMC3711841 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Replication of individual DNA molecules under electronic control using a protein nanopore.

Olasagasti Felix F Lieberman Kate R KR Benner Seico S Cherf Gerald M GM Dahl Joseph M JM Deamer David W DW Akeson Mark M

Nature nanotechnology 20100926 11

Nanopores can be used to analyse DNA by monitoring ion currents as individual strands are captured and driven through the pore in single file by an applied voltage. Here, we show that serial replication of individual DNA templates can be achieved by DNA polymerases held at the α-haemolysin nanopore orifice. Replication is blocked in the bulk phase, and is initiated only after the DNA is captured by the nanopore. We used this method, in concert with active voltage control, to observe DNA replicat ...[more]

PMID: 20871614

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Project description:The spatial organization of DNA is mediated by the Par protein system in some bacteria. ParB binds specifically to the parS sequence on DNA and orchestrates its motion by interacting with ParA bound to the nucleoid. In the case of plasmids, a single ParB bound plasmid is observed to execute oscillations between cell poles while multiple plasmids eventually settle at equal distances from each other along the cell's length. While the potential mechanism underlying the ParA-ParB interaction has been discussed, it remains unclear whether ParB-complex oscillations are stable limit cycles or merely decaying transients to a fixed point. How are dynamics affected by substrate length and the number of complexes? We present a deterministic model for ParA-ParB driven DNA segregation where the transition between stable arrangements and oscillatory behaviour depends only on five parameters: ParB-complex number, substrate length, ParA concentration, ParA hydrolysis rate and the ratio of the lengthscale over which the ParB complex stimulates ParA hydrolysis to the lengthscale over which ParA interacts with the ParB complex. When the system is buffered and the ParA rebinding rate is constant we find that ParB-complex dynamics is independent of substrate length and complex number above a minimum system size. Conversely, when ParA resources are limited, we find that changing substrate length and increasing complex number leads to counteracting mechanisms that can both generate or subdue oscillatory dynamics. We argue that cells may be poised near a critical level of ParA so that they can transition from oscillatory to fixed point dynamics as the cell cycle progresses so that they can both measure their size and faithfully partition their genetic material. Lastly, we show that by modifying the availability of ParA or depletion zone size, we can capture some of the observed differences in ParB-complex positioning between replicating chromosomes in B. subtilis cells and low-copy plasmids in E. coli cells.

Dataset Information

Replication of individual DNA molecules under electronic control using a protein nanopore.

Publications

Replication of individual DNA molecules under electronic control using a protein nanopore.

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