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

Abstract

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...

Single molecule techniques in DNA repair: A primer

“…As such, it degrades DNA ends until it recognizes a Chi sequence, when it stops degrading the 3′ strand but continues unwinding and degrading the 5′ strand, thereby creating a ssDNA overhang, which can be further processed to repair the DNA. This has recently been demonstrated at the single molecule level using magnetic tweezers [109]. In this study the authors observed pausing at Chi and Chi-like sequences and therefore propose a model for Chi recognition, whereby the enzyme pauses and undergoes a multistep conformational change involving the extrusion of a ssDNA loop, before resuming translocation after the Chi site [109].…”

“…Magnetic tweezers have been used to study topoisomerases [104], polymerases [105], restriction enzymes [64,106] and helicases involved in DNA repair [107–109] amongst others. One such study investigated UvrD unwinding rate, lifetime, and base pairs unwound.…”

“…This has recently been demonstrated at the single molecule level using magnetic tweezers [109]. In this study the authors observed pausing at Chi and Chi-like sequences and therefore propose a model for Chi recognition, whereby the enzyme pauses and undergoes a multistep conformational change involving the extrusion of a ssDNA loop, before resuming translocation after the Chi site [109]. This mechanism of target site search may be a general mechanism employed by many DNA binding proteins, whereby multiple weak binding events, to sites similar to the target, occur before a more stable protein-target complex is formed.…”

Bulk and single-molecule analysis of a novel DNA2-like helicase-nuclease reveals a single-stranded DNA looping motor

“…The quoted distances in base pairs were corrected using the value given by the worm-like chain model of rise per base pair of dsDNA at a given force. The unwinding rate was calculated by using the derivative of the smoothed data at 3 Hz in order to separate movement from pausing events (23).…”

The C-terminal domain of ParB is critical for dynamic DNA binding and bridging interactions which condense the bacterial centromere

“…We used a home-made magnetic tweezers setup similar in design to that described in (58,59) and detailed in (60). In brief, images of 1 μm superparamagnetic beads tethered to the surface of a glass slide by DNA constructs are acquired with a 100x oil immersion objective and a CCD camera.…”