The Bacillus subtilis AddAB helicase/nuclease is regulated by its cognate Chi sequence in vitro.
The AddAB enzyme is important to homologous DNA recombination in Bacillus subtilis, where it is thought to be the functional counterpart of the RecBCD enzyme of Escherichia coli. In vivo, AddAB responds to a specific five-nucleotide sequence (5'-AGCGG-3' or its complement) in a manner analogous to the response of the RecBCD enzyme to interaction with chi sequences. Here, we show that purified AddAB enzyme is able to load at a double-stranded DNA end and is both a DNA helicase and nuclease, whose combined action results in the degradation of both strands of the DNA duplex. During translocation, recognition of the properly oriented sequence 5'-AGCGG-3' causes attenuation of the AddAB enzyme nuclease activity that is responsible for degradation of the strand 3'-terminal at the entry site. Therefore, we conclude that 5'-AGCGG-3' is the B. subtilis Chi site and it is hereafter referred to as chi(Bs). After encountering chi(Bs), both the degradation of the 5'-terminal strand and the helicase activity persist. Thus, processing of a double-stranded DNA end by the AddAB enzyme produces a duplex DNA molecule with a protruding 3'-terminated single-stranded tail, a universal intermediate of the recombination process.
Chédin
F
F
Sections of Microbiology and of Molecular and Cellular Biology, University of California, Davis, CA 95616-8665, USA.
Ehrlich
S D
SD
Kowalczykowski
S C
SC
D001287:Attachment Sites, Microbiological / Q000235:genetics*; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D019143:Evolution, Molecular; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D005092:Exonucleases / Q000737:chemistry / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008970:Molecular Weight; D010957:Plasmids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D005090:Exodeoxyribonucleases; D005092:Exonucleases; C407424:AddAB enzyme; D043211:Exodeoxyribonuclease V; D004265:DNA Helicases; D008274:Magnesium