Translocation by the RecB motor is an absolute requirement for {chi}-recognition and RecA protein loading by RecBCD enzyme.
RecBCD enzyme is a heterotrimeric helicase/nuclease that initiates homologous recombination at double-stranded DNA breaks. The enzyme is driven by two motor subunits, RecB and RecD, translocating on opposite single-strands of the DNA duplex. Here we provide evidence that, although both motor subunits can support the translocation activity for the enzyme, the activity of the RecB subunit is necessary for proper function of the enzyme both in vivo and in vitro. We demonstrate that the RecBCD(K177Q) enzyme, in which RecD helicase is disabled by mutation of the ATPase active site, complements recBCD deletion in vivo and displays all of the enzymatic activities that are characteristic of the wild-type enzyme in vitro. These include helicase and nuclease activities and the abilities to recognize the recombination hotspot chi and to coordinate the loading of RecA protein onto the ssDNA it produces. In contrast, the RecB(K29Q)CD enzyme, carrying a mutation in the ATPase site of RecB helicase, fails to complement recBCD deletion in vivo. We further show that even though RecB(K29Q)CD enzyme displays helicase and nuclease activities, its inability to translocate along the 3'-terminated strand results in the failure to recognize chi and to load RecA protein. Our findings argue that translocation by the RecB motor is required to deliver RecC subunit to chi, whereas the RecD subunit has a dispensable motor activity but an indispensable regulatory function.
Spies
Maria
M
Sections of Microbiology and of Molecular and Cellular Biology, Center for Genetics and Development, University of California, Davis, California 95616, USA.
Dillingham
Mark S
MS
Kowalczykowski
Stephen C
SC
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D004265:DNA Helicases / Q000737:chemistry; D004269:DNA, Bacterial / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D043211:Exodeoxyribonuclease V / Q000737:chemistry / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D021381:Protein Transport; D011938:Rec A Recombinases / Q000737:chemistry / Q000378:metabolism*; D011995:Recombination, Genetic
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli; D000251:Adenosine Triphosphatases; D004265:DNA Helicases