Insights into Chi recognition from the structure of an AddAB-type helicase–nuclease complexHomologous recombination DNA repair requires double-strand break resection by helicase–nuclease enzymes. The crystal structure of bacterial AddAB in complex with DNA substrates shows that it employs an inactive helicase site to recognize ‘Chi' recombination hotspot sequences that regulate 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. AddAB was prone to stochastic pausing due to transient recognition of Chi-like sequences, unveiling an antagonistic relationship between DNA translocation and sequence-specific DNA recognition. Pauses at bona fide Chi sequences were longer, were nonexponentially distributed, and resulted in an altered velocity upon restart of translocation downstream of Chi. We propose a model for the recognition of Chi sequences to explain the origin of pausing during failed and successful hotspot recognition.protein motor | single molecule biophysics | DNA-end processing | real-time measurements | protein-DNA interactions D ouble-stranded DNA breaks (DSBs) are formed frequently, both as a result of exogenous and endogenous DNA damaging agents and as intermediates in programmed DNA rearrangements. Failure to properly repair DSBs results in loss of chromosome structural integrity and genomic instability and is associated with developmental defects, deficiencies of the immune system, and cancer predisposition. There are multiple mechanisms for DSB repair, but faithful repair generally requires the homologous recombination pathway, which can occur only if a suitable donor molecule such as the sister chromatid is available (1). Recombinational repair of DSBs is initiated by the longrange resection of the DNA end to form a 3′-terminated ssDNA overhang that acts as a substrate for the RecA/Rad51 recombinase. In all domains of life, this reaction is catalyzed by an array of helicases and nucleases but is best characterized in bacteria where either an AddAB-or a RecBCD-type helicase-nuclease complex recognizes the DNA end structure and then promotes its processive unwinding and concomitant resection (2, 3). A third class of helicase-nuclease named AdnAB is found in the mycobacterial niche and it shares limited structural similarity with AddAB enzymes (4). An apparently unique feature of the DNA break processing in bacteria is its control by cisacting DNA sequences called Crossover hotspot instigator (Chi) sequences. In the absence of hotspot sequences, AddAB or RecBCD complexes processively and rapidly degrade DNA in an ATP-dependent fashion, a mode of action that probably acts to degrade foreign DNA or in the restart of regressed replication forks (5). Recognition of Chi sequences by the translocating enzymes has many different effects on AddAB and/or RecBCD complexes, all of which serve to promote downstream recombination (6). These include the attenuation of the nuclease activity downstream of Chi on the 3′ strand (7), the promotion of DNA unwinding (8), and the lo...

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.

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. Binding of Chi to AddAB results in the attenuation of nuclease activity on the 3′-terminated strand, thereby promoting recombination. In this work, we used stopped-flow methods to monitor the coupling of adenosine triphosphate (ATP) hydrolysis and DNA translocation and how this is affected by Chi recognition. We show that in the absence of Chi sequences, AddAB translocates processively on DNA at ∼2000 bp s−1 and hydrolyses approximately 1 ATP molecule per base pair travelled. The recognition of recombination hotspots results in a sustained decrease in the translocation rate which is accompanied by a decrease in the ATP hydrolysis rate, such that the coupling between these activities and the net efficiency of DNA translocation is largely unchanged by Chi.

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. This transition is thought to be controlled by the Chi-dependent opening of a molecular latch, which enables part of the DNA substrate to evade degradation beyond Chi. Here, we show that disruption of the latch improves Chi recognition efficiency and stabilizes the interaction of AddAB with Chi, even in mutants that are impaired for Chi binding. Chi recognition elicits a structural change in AddAB that maps to a region of AddB which resembles a helicase domain, and which harbours both the Chi recognition locus and the latch. Mutation of the latch potentiates the change and moderately reduces the duration of a translocation pause at Chi. However, this mutant displays properties of Chi-modified AddAB even in the complete absence of bona fide hotspot sequences. The results are used to develop a model for AddAB regulation in which allosteric communication between Chi binding and latch opening ensures quality control during recombination hotspot recognition.