A dual-nuclease DNA break processing mechanism in AddAB enzyme
Yeeles, J. and Dillingham, M.S.
DNA-Protein interactions Unit, Dept. Biochemistry, Bristol University Medical School,
Bristol, BS8 1TD, UK.
Email: mark.dillingham@bristol.ac.uk
Nature has devised many strategies for repairing breaks in double-stranded DNA. In
bacterial homology-dependent pathways, the break is first processed to a 3-ssDNA
overhang at recombination hotspot (Chi) sequences1. This then serves as a substrate for
RecA-mediated strand exchange, allowing error-free repair of the broken DNA using a
homologous DNA duplex as a template. Here, we reveal a novel mechanism for DNA
break processing employed by the AddAB class of helicase-nuclease. We demonstrate that
the enzyme complex contains two active nuclease domains, each of which is dedicated for
the cleavage of one specific DNA strand. A nuclease activity responsible for processive
cleavage in the 3-5 direction is located in the AddA subunit, and is attenuated when the
enzyme complex encounters a recombination hotspot. In contrast, DNA cleavage in the 53 direction is attributable to the AddB subunit, and is unaffected by the encounter with a
recombination hotspot. Mutant proteins that are unable to cleave DNA in the 3-5 direction
cannot generate 3-ssDNA tails at recombination hotspots. However, these mutant
complexes retain the ability to recognise and form stable complexes with the Chi sequence,
demonstrating that the recognition and response to hotspot sequences can be uncoupled. A
model is presented for DNA break processing by AddAB enzyme using Chi-regulated dual
nuclease activities.
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1. Chedin, F and Kowalczykowski, S.C. (2002) A novel family of regulated helicases/nucleases from Grampositive bacteria: insights into the initiation of DNA recombination. Mol. Microbiol. 43, 823-834.