Identification of the nuclease active site in the multifunctional RecBCD enzyme by creation of a chimeric enzyme 1 1Edited by M. Gottesman

Yu, Misook; Souaya, Jehanne; Julin, Douglas A.

doi:10.1006/jmbi.1998.2127

Cited by 94 publications

(127 citation statements)

References 55 publications

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“…nificant nuclease activity (26). Furthermore, does not induce nuclease activity in either enzyme 3 (26). Thus, processing of linear DNA by either RecB D1080A CD or RecBC enzyme produces only full-length ssDNA.…”

Section: Figmentioning

confidence: 99%

“…Adapted from Anderson and Kowalczykowski (18). nificant nuclease activity (26). Furthermore, does not induce nuclease activity in either enzyme 3 (26).…”

Section: Figmentioning

confidence: 99%

“…indicates that the RecD subunit plays an essential role in DNA degradation. However, analysis of mutations in the RecB subunit establishes that the C-terminal domain of this enzyme is absolutely essential for all nuclease activities of the RecBCD enzyme (25,26). Furthermore, fusion of the C-terminal domain of the RecB subunit with the DNA binding domain of T4 phage gene 32 protein creates a nonspecific nuclease, showing that the C terminus of RecB subunit is indeed a nuclease.…”

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confidence: 99%

“…No contaminating protein bands were detected when 1 g of protein was loaded on an SDS-polyacrylamide gel and stained with Coomassie Brilliant Blue. RecB D1080A CD enzyme was a gift from D. A. Julin and M. Yu (University of Maryland) (26). E. coli SSB protein was isolated from strain RLM727 and purified according to LeBowitz (27).…”

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A Single Mutation, RecBD1080A, Eliminates RecA Protein Loading but Not Chi Recognition by RecBCD Enzyme

Anderson

Churchill

Kowalczykowski

1999

Journal of Biological Chemistry

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Homologous recombination and double-stranded DNA break repair in Escherichia coli are initiated by the multifunctional RecBCD enzyme. After binding to a double-stranded DNA end, the RecBCD enzyme unwinds and degrades the DNA processively. This processing is regulated by the recombination hot spot, Chi (: 5-GCT-GGTGG-3), which induces a switch in the polarity of DNA degradation and activates RecBCD enzyme to coordinate the loading of the DNA strand exchange protein, RecA, onto the single-stranded DNA products of unwinding. Recently, a single mutation in RecB, Asp-1080 3 Ala, was shown to create an enzyme (RecB D1080A CD) that is a processive helicase but not a nuclease. Here we show that the RecB D1080A CD enzyme is also unable to coordinate the loading of the RecA protein, regardless of whether sites are present in the DNA. However, the RecB D1080A CD enzyme does respond to sites by inactivating in a -dependent manner. These data define a locus of the RecBCD enzyme that is essential not only for nuclease function but also for the coordination of RecA protein loading.

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Section: Figmentioning

confidence: 99%

“…Adapted from Anderson and Kowalczykowski (18). nificant nuclease activity (26). Furthermore, does not induce nuclease activity in either enzyme 3 (26).…”

Section: Figmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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A Single Mutation, RecBD1080A, Eliminates RecA Protein Loading but Not Chi Recognition by RecBCD Enzyme

Anderson

Churchill

Kowalczykowski

1999

Journal of Biological Chemistry

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“…The individual activities of RecBCD enzyme depend on complex interactions between the three subunits, but at least part of the nuclease domain has been located in the C-terminal portion of the RecB polypeptide (26). A single bp change in recB resulting in the substitution of alanine (A) for aspartic acid (D) at position 1080 eliminates the nuclease activity and some of the Chi-dependent activities of the holoenzyme (27).…”

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confidence: 99%

The RecD subunit of the Escherichia coli RecBCD enzyme inhibits RecA loading, homologous recombination, and DNA repair

Amundsen

Taylor

Smith

2000

Proc. Natl. Acad. Sci. U.S.A.

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. 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...

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On the structure and operation of type I DNA restriction enzymes

Davies

Martin

Sturrock

et al. 1999

Journal of Molecular Biology

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Identification of the nuclease active site in the multifunctional RecBCD enzyme by creation of a chimeric enzyme 1 1Edited by M. Gottesman

Cited by 94 publications

References 55 publications

A Single Mutation, RecBD1080A, Eliminates RecA Protein Loading but Not Chi Recognition by RecBCD Enzyme

A Single Mutation, RecBD1080A, Eliminates RecA Protein Loading but Not Chi Recognition by RecBCD Enzyme

The RecD subunit of the Escherichia coli RecBCD enzyme inhibits RecA loading, homologous recombination, and DNA repair

On the structure and operation of type I DNA restriction enzymes

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