11 research outputs found
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
An Iron-Sulfur Cluster Is Essential for the Binding of Broken DNA by AddAB-type Helicase-Nucleases*S⃞
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