ࡱ> AC@ bjbj̚̚ 8>>"""" .$":<RRRRR---,v("-----RR-RR-:,}g#,X"!  0:+R""}} ------:"---------> :   A dual-nuclease DNA break processing mechanism in AddAB enzymeYeeles, J. and Dillingham, M.S.DNA-Protein interactions Unit, Dept. Biochemistry, Bristol University Medical School, Bristol, BS8 1TD, UK. Email: mark.dillingham@bristol.ac.ukNature 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. PLEASE CONFORM TO THIS STYLE - FIGURES MAY BE ADDED WITHIN THIS BOX AS REQUIRED THE FILE MUST CONSIST OF ONE A4 PAGE DO NOT CHANGE PAGE SET-UP SETTINGS1. Chedin, F and Kowalczykowski, S.C. (2002) A novel family of regulated helicases/nucleases from Gram-positive bacteria: insights into the initiation of DNA recombination. Mol. Microbiol. 43, 823-834.  >@JO_alm J K M N   r  ~h\?h.Z_HaJmH sH h\?h.Z5_HaJh\?h.Z6_HaJh\?h.Z_HaJhRzh.Z_H *h.Z *h\?h.Zh\?h.ZH* jh\?h.Z h)h.Z h/Vh.Zh\?h.Z>* hnh.Z hRzh.Zh.Z*?@` $Ifgd.Zekd$$Ifl"y$ t0644 la $Ifgd.Z`a/ekd$$Ifl"y$ t0644 la$Ifekdo$$Ifl"y$ t0644 lar   $Ifgd.ZekdM$$Ifl"y$ t0644 la $Ifgd.Zgkd$$Ifl"y$ t0644 la6&P 1F:p.Z. 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