Specific nucleotide binding and rebinding to individual DNA polymerase complexes captured on a nanopore.

Nanoscale pores are a tool for single molecule analysis of DNA or RNA processing enzymes. Monitoring catalytic activity in real time using this technique requires that these enzymes retain function while held atop a nanopore in an applied electric field. Using an alpha-hemolysin nanopore, we measured the dwell time for complexes of DNA with the Klenow fragment of Escherichia coli DNA polymerase I (KF) as a function of the concentration of deoxynucleoside triphosphate (dNTP) substrate. We analyzed these dwell time measurements in the framework of a two-state model for captured complexes (DNA-KF binary and DNA-KF-dNTP ternary states). Average nanopore dwell time increased without saturating as a function of correct dNTP concentration across 4 orders of magnitude. This arises from two factors that are proportional to dNTP concentration: (1) The fraction of complexes that are in the ternary state when initially captured predominantly affects dwell time at low dNTP concentrations. (2) The rate of binding and rebinding of dNTP to captured complexes affects dwell time at higher dNTP concentrations. Thus there are two regimes that display a linear relationship between average dwell time and dNTP concentration. The transition from one linear regime to the other occurs near the equilibrium dissociation constant (K(d)) for dNTP binding to KF-DNA complexes in solution. We conclude from the combination of titration experiments and modeling that DNA-KF complexes captured atop the nanopore retain iterative, sequence-specific dNTP binding, as required for catalysis and fidelity in DNA synthesis.

Measuring and modeling the kinetics of individual DNA-DNA polymerase complexes on a nanopore.

The assembly of a DNA-DNA polymerase binary complex is the precursory step in genome replication, in which the enzyme binds to the 3' junction created when a primer binds to its complementary substrate. In this study, we use an active control method for observing the binding interaction between Klenow fragment (exo-) (KF) in the bulk-phase chamber above an α-hemolysin (α-HL) nanopore and a single DNA molecule tethered noncovalently in the nanopore. Specifically, the control method regulates the temporal availability of the primer-template DNA to KF binding and unbinding above the nanopore, on millisecond-to-second time scales. Our nanopore measurements support a model that incorporates two mutually exclusive binding states of KF to DNA at the primer-template junction site, termed "weakly bound" and "strongly bound" states. The composite binding affinity constant, the equilibrium constant between the weak and strong states, and the unbound-to-strong association rate are quantified from the data using derived modeling analysis. The results support that the strong state is in the nucleotide incorporation pathway, consistent with other nanopore assays. Surprisingly, the measured unbound-to-strong association process does not fit a model that admits binding of only free (unbound) KF to the tethered DNA but does fit an association rate that is proportional to the total (unbound and DNA-bound) KF concentration in the chamber above the nanopore. Our method provides a tool for measuring pre-equilibrium kinetics one molecule at a time, serially and for tens of thousands of single-molecule events, and can be used for other polynucleotide-binding enzymes.

Sequence-specific detection of individual DNA polymerase complexes in real time using a nanopore.

Nanoscale pores have potential to be used as biosensors and are an established tool for analysing the structure and composition of single DNA or RNA molecules. Recently, nanopores have been used to measure the binding of enzymes to their DNA substrates. In this technique, a polynucleotide bound to an enzyme is drawn into the nanopore by an applied voltage. The force exerted on the charged backbone of the polynucleotide by the electric field is used to examine the enzyme-polynucleotide interactions. Here we show that a nanopore sensor can accurately identify DNA templates bound in the catalytic site of individual DNA polymerase molecules. Discrimination among unbound DNA, binary DNA/polymerase complexes, and ternary DNA/polymerase/deoxynucleotide triphosphate complexes was achieved in real time using finite state machine logic. This technique is applicable to numerous enzymes that bind or modify DNA or RNA including exonucleases, kinases and other polymerases.

The host resistance locus sst1 controls innate immunity to Listeria monocytogenes infection in immunodeficient mice.

Epidemiological, clinical, and experimental approaches have convincingly demonstrated that host resistance to infection with intracellular pathogens is significantly influenced by genetic polymorphisms. Using a mouse model of infection with virulent Mycobacterium tuberculosis (MTB), we have previously identified the sst1 locus as a genetic determinant of host resistance to tuberculosis. In this study we demonstrate that susceptibility to another intracellular pathogen, Listeria monocytogenes, is also influenced by the sst1 locus. The contribution of sst1 to anti-listerial immunity is much greater in immunodeficient scid mice, indicating that this locus controls innate immunity and becomes particularly important when adaptive immunity is significantly depressed. Similar to our previous observations using infection with MTB, the resistant allele of sst1 prevents formation of necrotic infectious lesions in vivo. We have shown that macrophages obtained from sst1-resistant congenic mice possess superior ability to kill L. monocytogenes in vitro. The bactericidal effect of sst1 is dependent on IFN-gamma activation and reactive oxygen radical production by activated macrophages after infection, but is independent of NO production. It is possible that there is a single gene that controls common IFN-dependent macrophage function, which is important in the pathogenesis of infections caused by both MTB and L. monocytogenes. However, host resistance to the two pathogens may be controlled by two different polymorphic genes encoded within the sst1 locus. The polymorphic gene(s) encoded within the sst1 locus that controls macrophage interactions with the two intracellular pathogens remains to be elucidated.