We have developed and optimized a stopped-flow fluorescence assay for use in studying DNA unwinding catalyzed by Escherichia coli RecBCD helicase. This assay monitors changes in fluorescence resonance energy transfer (FRET) between a pair of fluorescent probes (Cy3 donor and Cy5 acceptor) placed on opposite sides of a nick in duplex DNA. As such, this is an "all-or-none" DNA unwinding assay. Si...

Helicase-catalyzed DNA unwinding is often studied using "all or none" assays that detect only the final product of fully unwound DNA. Even using these assays, quantitative analysis of DNA unwinding time courses for DNA duplexes of different lengths, L, using "n-step" sequential mechanisms, can reveal information about the number of intermediates in the unwinding reaction and the "kinetic step s...

The abilities of N-acetoxy-2-acetylaminofluorene and a series of hydrocarbon epoxides to cause the unwinding of supercoiled SV40 DNA by covalent binding were mea sured by incubating the DNA with the compound at pH 7.4 at 37° and then determining the superhelicity of the modi fied DNA by gel electrophoresis. The concentrations of the electrophiles required to unwind fully the supercoiled DNA ra...

Replicative helicases are essential molecular machines that utilize energy derived from NTP hydrolysis to move along nucleic acids and to unwind double-stranded DNA (dsDNA). Our earlier crystal structure of the hexameric helicase from Geobacillus kaustophilus HTA426 (GkDnaC) in complex with single-stranded DNA (ssDNA) suggested several key residues responsible for DNA binding that likely play a...

Geminivirus replication initiator protein (Rep) is a multifunctional viral required for replication. During the process of replication, Rep acts as site- and strand-specific endonuclease, ligase, ATPase, helicase. B' motif β-hairpin loop geminivirus are conserved important Rep-mediated helicase activity To dissect roles various amino acid residues in its unwinding DNA, we investigated eight nea...

The minichromosome maintenance (MCM) helicase complex is essential for the initiation and elongation of DNA replication in both the eukaryotic and archaeal domains. The archaeal homohexameric MCM helicase from Sulfolobus solfataricus serves as a model for understanding mechanisms of DNA unwinding. In this report, the displaced 5'-tail is shown to provide stability to the MCM complex on DNA and ...

All cellular single-stranded (ss) DNA is rapidly bound and stabilized by single stranded DNA-binding proteins (SSBs). Replication protein A, the main eukaryotic SSB, is able to unwind double-stranded (ds) DNA by binding and stabilizing transiently forming bubbles of ssDNA. Here, we study the dynamics of human RPA (hRPA) activity on topologically constrained dsDNA with single-molecule magnetic t...

The adenovirus (Ad) DNA-binding protein (DBP) is essential for the elongation phase of Ad DNA replication by unwinding the template in an ATP-independent fashion, employing its capacity to form multimers. DBP also enhances the rate of initiation, with the highest levels obtained at low concentrations of Ad DNA polymerase (Pol). Here, we show that stimulation of initiation depends on the templat...