Helicases are enzymes that couple the hydrolysis of ATP to the unwinding of duplex nucleic acids (NAs), thus providing the single-stranded NA (ssNA) intermediates necessary for nucleic acid processing and maintenance such as replication, recombination, repair, and transcription (1–3). All helicases possess a core helicase domain containing RecA-like motifs that are responsible for NA binding an...

The Escherichia coli UvrD helicase is known to function in the mismatch repair and nucleotide excision repair pathways and has also been suggested to have roles in recombination and replication restart. The primary intermediate DNA structure in these two processes is the Holliday junction. UvrD has been shown to unwind a variety of substrates including partial duplex DNA, nicked DNA, forked DNA...

A simple model for the force-dependent unwinding and rewinding rates of the nucleosome inner turn is constructed and quantitatively compared to the results of recent measurements [A. H. Mack et al., J. Mol. Biol. 423, 687 (2012)]. First, a coarse-grained model for the histone-DNA free-energy landscape that incorporates both an elastic free-energy barrier and specific histone-DNA bonds is develo...

Replication in vitro of the replicative form (RF) I DNA of bacteriophage varphiX174 requires the phage-induced cistron A (cisA) protein, the host rep protein, DNA-binding protein, ATP, and DNA polymerase III plus replication factors. The rep protein is a single-stranded DNA-dependent ATPase. In this paper we show that varphiX174 RF I DNA cut by the cisA protein acts as a duplex DNA cofactor for...

We describe an assay to measure the extent of enzymatic unwinding of DNA by a DNA helicase. This assay takes advantage of the quenching of the intrinsic protein fluorescence of Escherichia coli SSB protein upon binding to ssDNA and is used to characterize the DNA unwinding activity of recBCD enzyme. Unwinding in this assay is dependent on the presence of recBCD enzyme and linear dsDNA, is consi...

Herpes simplex virus 1 contains three origins of replication; two copies of oriS and one of a similar sequence, oriL. Here, the combined action of multiple factors known or thought to influence the opening of oriS are examined. These include the viral origin-binding protein, UL9, and single-strand binding protein ICP8, host cell topoisomerase I, and superhelicity of the DNA template. By using e...

RecBCD is a multifunctional enzyme that possesses both helicase and nuclease activities. To gain insight into the mechanism of its helicase function, RecBCD unwinding at low adenosine triphosphate (ATP) (2-4 μM) was measured using an optical-trapping assay featuring 1 base-pair (bp) precision. Instead of uniformly sized steps, we observed forward motion convolved with rapid, large-scale (∼4 bp)...

Hexameric helicases control both the initiation and the elongation phase of DNA replication. The toroidal structure of these enzymes provides an inherent challenge in the opening and loading onto DNA at origins, as well as the conformational changes required to exclude one strand from the central channel and activate DNA unwinding. Recently, high-resolution structures have not only revealed the...

Antitumor cisplatin [cis-diamminedichloroplatinum(II)] forms on DNA predominantly intrastrand cross-links between neighboring purine residues. Several discoveries suggested that the toxicity of cisplatin originated from these lesions. The formation of 1,2-GG intrastrand cross-link of cisplatin leads to marked conformational alterations in DNA including a directional, rigid bend toward the major...

RecQ helicases have essential roles in maintaining genome stability during replication and in controlling double-strand break repair by homologous recombination. Little is known about how the different RecQ helicases found in higher eukaryotes achieve their specialized and partially opposing functions. Here, we investigate the DNA unwinding of RecQ helicases from Arabidopsis thaliana, AtRECQ2 a...