Replication Dynamics

Abstract:

Using a combination of single molecule and bulk solution measurements, we have examined the DNA translocation activity of a helicase, the Type I restriction modification enzyme EcoR124I. We find that EcoR124I can translocate past covalent interstrand crosslinks, inconsistent with an obligatory unwinding mechanism. Instead, translocation of the intact dsDNA occurs principally via contacts to the sugar-phosphate backbone and bases of the 3'-5' strand; contacts to the 5'-3' strand are not essential for motion but do play a key role in stabilising the motor on the DNA. A model for dsDNA translocation is presented that could be applicable to a wide range of other enzyme complexes that are also labelled as helicases but which do not have actual unwinding activity.

Abstract:

Most analyses of single-molecule experiments consist of binning experimental outcomes into a histogram and finding the parameters that optimize the fit of this histogram to a given data model. Here we show that such an approach can introduce biases in the estimation of the parameters, thus great care must be taken in the estimation of model parameters from the experimental data. The bias can be particularly large when the observations themselves are not statistically independent and are subjected to global constraints, as, for example, when the iterated steps of a motor protein acting on a single molecule must not exceed the total molecule length. We have developed a maximum-likelihood analysis, respecting the experimental constraints, which allows for a robust and unbiased estimation of the parameters, even when the bias well exceeds 100%. We demonstrate the potential of the method for a number of single-molecule experiments, focusing on the removal of DNA supercoils by topoisomerase IB, and validate the method by numerical simulation of the experiment.

Abstract:

We report experimental measurements of the salt dependence of ion transport and DNA translocation through solid-state nanopores. The ionic conductance shows a three-order-of-magnitude decrease with decreasing salt concentrations from 1 M to 1 muM, strongly deviating from bulk linear behavior. The data are described by a model that accounts for a salt-dependent surface charge of the pore. Subsequently, we measure translocation of 16.5-mum-long dsDNA for 50 mM to 1 M salt concentrations. DNA translocation is shown to result in either a decrease ([KCl] > 0.4 M) or increase of the ionic current ([KCl] < 0.4 M). The data are described by a model where current decreases result from the partial blocking of the pore and current increases are attributed to motion of the counterions that screen the charge of the DNA backbone. We demonstrate that the two competing effects cancel at a KCl concentration of 370 +/- 40 mM.

Abstract:

Several bioinformatics studies have identified an unexpected but remarkably prevalent approximately 10 bp periodicity of AA/TT dinucleotides (hyperperiodicity) in certain regions of the Caenorhabditis elegans genome. Although the relevant C.elegans DNA segments share certain sequence characteristics with bent DNAs from other sources (e.g. trypanosome mitochondria), the nematode sequences exhibit a much more extensive and defined hyperperiodicity. Given the presence of hyperperiodic structures in a number of critical C.elegans genes, the physical characteristics of hyperperiodic DNA are of considerable interest. In this work, we demonstrate that several hyperperiodic DNA segments from C.elegans exhibit structural anomalies using high-resolution atomic force microscopy (AFM) and gel electrophoresis. Our quantitative analysis of AFM images reveals that hyperperiodic DNA adopts a significantly smaller mean square end-to-end distance, hence a more compact coil structure, compared with non-periodic DNA of similar length. While molecules remain capable of adopting both bent and straight (rod-like) configurations, indicating that their flexibility is still retained, examination of the local curvatures along the DNA contour length reveals that the decreased mean square end-to-end distance can be attributed to the presence of long-scale intrinsic bending in hyperperiodic DNA. Such bending is not detected in non-periodic DNA. Similar studies of shorter, nucleosome-length DNAs that survived micrococcal nuclease digestion show that sequence hyperperiodicity in short segments can likewise induce strong intrinsic bending. It appears, therefore, that regions of the C.elegans genome display a significant correlation between DNA sequence and unusual mechanical properties.