Specific anchoring of large topologically closed DNA for single-molecule protein:DNA interactions.

Single-molecule and bulk biophysical approaches to study protein-DNA interactions on surface-immobilized nucleic acid templates typically rely on modifying the ends of linear DNA molecules to enable surface-DNA attachments. Unless both strands are constrained, this results in topologically free DNA molecules and the inability to observe supercoiling-dependent biological processes or requires additional means to micromanipulate the free DNA end to impose rotational constraints or induce supercoiling. We developed a method using RecA protein to induce the formation of a circularized compliment-stabilized D-loop.

Direct visualization of translesion DNA synthesis polymerase IV at the replisome.

In bacterial cells, DNA damage tolerance is manifested by the action of translesion DNA polymerases that can synthesize DNA across template lesions that typically block the replicative DNA polymerase III. It has been suggested that one of these translesion DNA synthesis DNA polymerases, DNA polymerase IV, can either act in concert with the replisome, switching places on the β sliding clamp with DNA polymerase III to bypass the template damage, or act subsequent to the replisome skipping over the template lesion in the gap in nascent DNA left behind as the replisome continues downstream. Evidence exists in support of both mechanisms.

Chi hotspots trigger a conformational change in the helicase-like domain of AddAB to activate homologous recombination.

In bacteria, the repair of double-stranded DNA breaks is modulated by Chi sequences. These are recognised by helicase-nuclease complexes that process DNA ends for homologous recombination. Chi activates recombination by changing the biochemical properties of the helicase-nuclease, transforming it from a destructive exonuclease into a recombination-promoting repair enzyme.

Probing DNA helicase kinetics with temperature-controlled magnetic tweezers.

Motor protein functions like adenosine triphosphate (ATP) hydrolysis or translocation along molecular substrates take place at nanometric scales and consequently depend on the amount of available thermal energy. The associated rates can hence be investigated by actively varying the temperature conditions. In this article, a thermally controlled magnetic tweezers (MT) system for single-molecule experiments at up to 40 °C is presented.

Engineering a reagentless biosensor for single-stranded DNA to measure real-time helicase activity in Bacillus.

Single-stranded DNA-binding protein (SSB) is a well characterized ubiquitous and essential bacterial protein involved in almost all aspects of DNA metabolism. Using the Bacillus subtilis SSB we have generated a reagentless SSB biosensor that can be used as a helicase probe in B. subtilis and closely related gram positive bacteria.

Recombination hotspots attenuate the coupled ATPase and translocase activities of an AddAB-type helicase-nuclease.

In all domains of life, the resection of double-stranded DNA breaks to form long 3'-ssDNA overhangs in preparation for recombinational repair is catalyzed by the coordinated activities of DNA helicases and nucleases. In bacterial cells, this resection reaction is modulated by the recombination hotspot sequence Chi. The Chi sequence is recognized in cis by translocating helicase-nuclease complexes such as the Bacillus subtilis AddAB complex.

On the mechanism of recombination hotspot scanning during double-stranded DNA break resection.

Double-stranded DNA break repair by homologous recombination is initiated by resection of free DNA ends to produce a 3'-ssDNA overhang. In bacteria, this reaction is catalyzed by helicase-nuclease complexes such as AddAB in a manner regulated by specific recombination hotspot sequences called Crossover hotspot instigator (Chi). We have used magnetic tweezers to investigate the dynamics of AddAB translocation and hotspot scanning during double-stranded DNA break resection.

Superfamily 1 helicases .

Superfamily 1 helicases are nucleic acid motor proteins that couple ATP hydrolysis to translocation along, and concomitant unwinding of, DNA or RNA. This is central to many aspects of cellular DNA and RNA metabolism and, accordingly, they are implicated in a wide range of nucleic acid processing events including DNA replication, recombination and repair as well as many aspects of RNA metabolism. This review discusses our current understanding of the structure, function and mechanism of Superfamily 1 helicases.

Insights into Chi recognition from the structure of an AddAB-type helicase-nuclease complex.

In bacterial cells, processing of double-stranded DNA breaks for repair by homologous recombination is dependent upon the recombination hotspot sequence Chi and is catalysed by either an AddAB- or RecBCD-type helicase-nuclease. Here, we report the crystal structure of AddAB bound to DNA. The structure allows identification of a putative Chi-recognition site in an inactivated helicase domain of the AddB subunit.