Joseph T.P. Yeeles

An Iron-Sulfur Cluster Is Essential for the Binding of Broken DNA by AddAB-type Helicase-Nucleases

The bacterial helicase-nuclease complex AddAB converts double-stranded DNA breaks into substrates for RecA-dependent recombinational repair. Here we show that the AddB subunit contains a novel class of nuclease domain distinguished by the presence of an iron-sulfur cluster. The cluster is coordinated by an unusual arrangement of cysteine residues that originate from both sides of the AddB nuclease, forming an “iron staple” that is required for the local structural integrity of this domain. Disruption of the iron-sulfur cluster by mutagenesis eliminates the ability of AddAB to bind to duplex DNA ends without affecting the single-stranded DNA-dependent ATPase activity. Sequence analysis suggests that a related iron staple nuclease domain is present in the eukaryotic DNA replication/repair factor Dna2, where it is also associated with a DNA helicase motor.

The processing of double-stranded DNA breaks for recombinational repair by helicase-nuclease complexes.

Double-stranded DNA breaks are prepared for recombinational repair by nucleolytic digestion to form single-stranded DNA overhangs that are substrates for RecA/Rad51-mediated strand exchange. This processing can be achieved through the activities of multiple helicases and nucleases. In bacteria, the function is mainly provided by a stable multi-protein complex of which there are two structural classes; AddAB- and RecBCD-type enzymes. These helicase-nucleases are of special interest with respect to DNA helicase mechanism because they are exceptionally powerful DNA translocation motors, and because they serve as model systems for both single molecule studies and for understanding how DNA helicases can be coupled to other protein machinery. This review discusses recent developments in our understanding of the AddAB and RecBCD complexes, focussing on their distinctive strategies for processing DNA ends. We also discuss the extent to which bacterial DNA end resection mechanisms may parallel those used in eukaryotic cells.

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. By generating mutant protein complexes that do not respond to Chi, we show that residues responsible for Chi recognition are located in positions equivalent to the signature motifs of a conventional helicase. Comparison with the related RecBCD complex, which recognizes a different Chi sequence, provides further insight into the structural basis for sequence‐specific ssDNA recognition. The structure suggests a simple mechanism for DNA break processing, explains how AddAB and RecBCD can accomplish the same overall reaction with different sets of functional modules and reveals details of the role of an Fe–S cluster in protein stability and DNA binding.

Visualizing helicases unwinding DNA at the single molecule level

DNA helicases are motor proteins that catalyze the unwinding of double-stranded DNA into single-stranded DNA using the free energy from ATP hydrolysis. Single molecule approaches enable us to address detailed mechanistic questions about how such enzymes move processively along DNA. Here, an optical method has been developed to follow the unwinding of multiple DNA molecules simultaneously in real time. This was achieved by measuring the accumulation of fluorescent single-stranded DNA-binding protein on the single-stranded DNA product of the helicase, using total internal reflection fluorescence microscopy. By immobilizing either the DNA or helicase, localized increase in fluorescence provides information about the rate of unwinding and the processivity of individual enzymes. In addition, it reveals details of the unwinding process, such as pauses and bursts of activity. The generic and versatile nature of the assay makes it applicable to a variety of DNA helicases and DNA templates. The method is an important addition to the single-molecule toolbox available for studying DNA processing enzymes.

The AddAB helicase–nuclease catalyses rapid and processive DNA unwinding using a single Superfamily 1A motor domain

The oligomeric state of Superfamily I DNA helicases is the subject of considerable and ongoing debate. While models based on crystal structures imply that a single helicase core domain is sufficient for DNA unwinding activity, biochemical data from several related enzymes suggest that a higher order oligomeric species is required. In this work we characterize the helicase activity of the AddAB helicase–nuclease, which is involved in the repair of double-stranded DNA breaks in Bacillus subtilis. We show that the enzyme is functional as a heterodimer of the AddA and AddB subunits, that it is a rapid and processive DNA helicase, and that it catalyses DNA unwinding using one single-stranded DNA motor of 3′→5′ polarity located in the AddA subunit. The AddB subunit contains a second putative ATP-binding pocket, but this does not contribute to the observed helicase activity and may instead be involved in the recognition of recombination hotspot sequences.

Recombination Hotspots and Single-Stranded DNA Binding Proteins Couple DNA Translocation to DNA Unwinding by the AddAB Helicase-Nuclease

AddAB is a helicase-nuclease that processes double-stranded DNA breaks for repair by homologous recombination. This process is modulated by Chi recombination hotspots: specific DNA sequences that attenuate the nuclease activity of the translocating AddAB complex to promote downstream recombination. Using a combination of kinetic and imaging techniques, we show that AddAB translocation is not coupled to DNA unwinding in the absence of single-stranded DNA binding proteins because nascent single-stranded DNA immediately re-anneals behind the moving enzyme. However, recognition of recombination hotspot sequences during translocation activates unwinding by coupling these activities, thereby ensuring the downstream formation of single-stranded DNA that is required for RecA-mediated recombinational repair. In addition to their implications for the mechanism of double-stranded DNA break repair, these observations may affect our implementation and interpretation of helicase assays and our understanding of helicase mechanisms in general.

Recombination Hotspots and SSB Proteins Couple Translocation and Unwinding Activities of the AddAb Helicase-Nuclease

Recombinational repair of DNA breaks requires processing of a DNA end to a 3′-ssDNA overhang. In B.subtilis, this task is done by the helicase-nuclease AddAB which generates ssDNA overhangs terminated at a recombination hotspot (Chi) sequence. In this work, we have used stopped flow DNA unwinding assays and atomic force microscopy to investigate the processing of DNA breaks by the AddAB helicase-nuclease. In the absence of single-stranded binding proteins, we found that translocation and unwinding activities of AddAB are uncoupled due to re-annealing of nascent single-stranded DNA. However, recognition of Chi sequences during AddAB translocation activates unwinding by coupling both activities. Helicase activity of AddAB is also activated by binding of SSB proteins or activity of multiple AddAB in multiple turnover reactions by preventing re-annealing of DNA strands. The implications of these findings for our understanding of DNA break repair intermediates and of general helicase mechanisms will be discussed.