The RecBCD enzyme of Escherichia coli promotes recombination preferentially at X nucleotide sequences and has in vivo helicase and strong duplex DNA exonuclease (exoV) activities. The enzyme without the RecD subunit, as in a recD null mutant, promotes recombination efficiently but independently of X and has no nucleolytic activity. Employing phage A red gam crosses, phage T4 2-survival measurements, and exoV assays, it is shown that E. coli cells in which RecBCD has extensive opportunity to interact with linear X-containing DNA (produced by rolling circle replication of a plasmid with X or by bleomycin-induced fragmentation of the cellular chromosome) acquire the phenotype of a recD mutant and maintain this for -2 h. It is concluded that RecBCD is converted into RecBC during interaction with X by irreversible inactivation of RecD. After conversion, the enzyme is released and initiates recombination on other DNA molecules in a X-independent fashion. Overexpression of recD+ (from a plasmid) prevented the phenotypic change and providing RecD after the change restored x-stimulated recombination. The observed recA+ dependence of the downregulation of exoV could explain the previously noted "reckless" DNA degradation of recA mutants. It is proposed that X sites are regulatory elements for the RecBCD to RecBC switch and thereby function as cis-and trans-acting stimulators of RecBC-dependent recombination.The RecBCD enzyme is an essential component of the main homologous recombination pathway in Escherichia coli (see refs. 1 and 2). This pathway recombines linear DNA molecules during conjugation, transduction, and vegetative phage crosses and is required for repair of double-strand breaks (3, 4). RecBCD enzyme is an ATP-dependent exonuclease (exoV) that consists of three protein subunits encoded by the recB, recC, and recD genes. ExoV activity is composed of ATPdependent DNA helicase and single-stranded DNA endonuclease activities of RecBCD enzyme acting on linear doublestranded DNA to produce single-stranded DNA oligonucleotides (see ref. 5). In cells and in cell-free extracts with ATP, RecBCD provides the major linear duplex DNA-degrading activity (6-8).X hot spots of RecBCD-dependent recombination are present in the E. coli genome -1000 times and stimulate, when present in phage A, recombination in A red gam crosses with decreasing efficiency in increasing distance on the left side of their sequence 5'-GCTGGTGG-3' as written here (see ref. 9). Hot spot activity is exerted only when RecBCD enters the DNA molecule from a double-stranded end placed on the 3' side of X (see ref. 9). An important finding was that recD null mutants were recombination proficient and as UV-resistant as wild-type cells (10, 11). They had lost all nucleolytic activities of RecBCD, and recombination was x independent (10, 11).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.Moreo...
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