Exonuclease V (EC 3.1.11.5) of Escherichia coli, an enzyme with multiple activities promoting genetic recombination, has previously been shown to contain two polypeptides, the products of the recB and recC genes. We report here that the enzyme contains in addition a third polypeptide (a) with a molecular mass of about 58 kDa. The a poypeptide is not synthesized by a class of mutants (previously designated recB*) lacking the nuclease activity of exonuclease V but retaining recombination proficiency. The gene, recD, coding for the a polypeptide is located near recB in the order thyA-recC-ptr-recB-recD-agA on the E. coUl chromosome. The recB and recD genes appear to be governed by a common promoter to the left of recB; a weaker promoter appears to govern recD alone. In the light of these results we discuss the relation between the structure and function of the three polypeptides of exonuclease V, hereby alternatively designated RecBCD enzyme.

SummaryHelicobacter pylori colonization of the human stomach is characterized by profound diseasecausing inflammation. Bacterial proteins that detoxify reactive oxygen species or recognize damaged DNA adducts promote infection, suggesting that H. pylori requires DNA damage repair for successful in vivo colonization. The molecular mechanisms of repair remain unknown. We identified homologues of the AddAB class of helicase-nuclease enzymes, related to the Escherichia coli RecBCD enzyme, which, with RecA, is required for repair of DNA breaks and homologous recombination. H. pylori mutants lacking addA or addB genes lack detectable ATPdependent nuclease activity, and the cloned H. pylori addAB genes restore both nuclease and helicase activities to an E. coli recBCD deletion mutant. H. pylori addAB and recA mutants have a reduced capacity for stomach colonization. These mutants are sensitive to DNA damaging agents and have reduced frequencies of apparent gene conversion between homologous genes encoding outer membrane proteins. Our results reveal requirements for doublestrand break repair and recombination during both acute and chronic phases of H. pylori stomach infection.

In Escherichia coli, at least two groups of proteins, or "recombination machines," can operate independently on broken DNA to produce a 3'-terminated single-stranded DNA filament coated with RecA protein and ready for synapsis with intact homologous DNA. Recent analyses of mutants lacking one or more of the activities required for presynaptic filament formation by one recombination machine demonstrate that parts of the two normally separate machines can interchange to initiate homologous recombination.

. We tested the hypothesis that the RecD subunit inhibits recombination by deleting recD from the nuclease-and recombination-deficient mutant recB D1080A CD. We report here that the resulting strain, recB D1080A C, was proficient for recombination and DNA repair. Recombination proficiency was accompanied by a change in enzyme activity: RecB D1080A C enzyme loaded RecA protein onto DNA during DNA unwinding whereas RecB D1080A CD enzyme did not. Together, these genetic and biochemical results demonstrate that RecA loading by RecBCD enzyme is required for recombination in E. coli cells and suggest that RecD interferes with the enzyme domain required for its loading. A nuclease-dependent signal appears to be required for a change in RecD that allows RecA loading. Because RecA loading is not observed with wild-type RecBCD enzyme until it acts at a Chi site, our observations support the view that RecD inhibits recombination until the enzyme acts at Chi. R ecBCD enzyme plays a central role in the major pathway of genetic exchange and DNA repair in Escherichia coli (1-3). The degradative and recombinational activities of RecBCD enzyme, as well as its structure, are regulated by a specific DNA sequence called Chi (5Ј-GCTGGTGG-3Ј). As a consequence of the RecBCD enzyme-Chi interaction, both the DNA substrate (4-7) and enzyme (8-10) are changed. Genetic and biochemical experiments have suggested that one or another RecBCD enzyme subunit directs this regulation (8,(11)(12)(13)(14)(15)(16). We show here that the RecD subunit inhibits E. coli recombination by blocking RecBCD enzyme-facilitated loading of RecA protein onto single-stranded (ss) DNA.The structure of the RecBCD enzyme and the activities it promotes are complex. RecBCD enzyme is a heterotrimer composed of one copy of each of the products of the recB, recC, and recD genes (17). The enzyme is an ATP-dependent doublestranded (ds) and ss exonuclease, a ss endonuclease, and a DNA helicase (1). RecBCD enzyme interacts with Chi sites, which stimulate recombination in E. coli and bacteriophage lambda (18). Null mutations in recB and recC result in recombination deficiency and sensitivity to DNA damaging agents (19)(20)(21). Cultures of such strains contain many inviable cells (22), reflecting their inability to repair DNA damage by homologous recombination. In contrast, null mutations in recD leave strains highly viable and proficient in recombination and DNA repair (refs. 11 and 23; see below).The role of RecBCD enzyme in homologous recombination begins when it binds to the end of a dsDNA substrate and initiates unwinding. Further reactions of RecBCD enzyme with DNA occur in a manner dependent on the presence of Chi sites and the concentrations of Mg 2ϩ and ATP. When the concentration of ATP exceeds that of Mg 2ϩ , RecBCD enzyme makes a ss endonucleolytic cut a few nt to the 3Ј side of Chi (4, 5). When the concentration of Mg 2ϩ exceeds that of ATP, RecBCD enzyme degrades the 3Ј terminated strand during DNA unwinding; the degradation is reduced when the enzyme reaches Ch...