The RecBC enzyme loads RecA protein onto ssDNA asymmetrically and independently of chi , resulting in constitutive recombination activation

Churchill, Jason J.; Anderson, Daniel G.; Kowalczykowski, Stephen C.

doi:10.1101/gad.13.7.901

Cited by 145 publications

(129 citation statements)

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“…This model is supported by the fact that recD mutants are recombination-proficient (11,23,29) and hyperre-combination-proficient in the absence of Chi sites (11), and they cluster recombination events at the ends of DNA in lambda replication-blocked crosses (30). Purified RecBC enzyme (i.e., lacking RecD) has a low affinity for dsDNA (unpublished data, see Table 5) ends but can unwind DNA (16,31,32) and facilitates loading of RecA protein at the 3Ј termini of DNA during unwinding (16). This is in contrast to RecBCD enzyme, which requires a Chi site for significant loading of RecA protein (6), joint molecule formation (7), and recombination (18).…”

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confidence: 71%

“…As a consequence of the RecBCD enzyme-Chi interaction, both the DNA substrate (4-7) and enzyme (8)(9)(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.…”

mentioning

confidence: 79%

“…2, lanes 8-10, Table 4). Mutant RecBCD enzymes that have unwinding activity but lack nuclease activity produce a full-length ss product and may load RecA constitutively on the 3Ј end (16). Thus, RecBC enzyme, which lacks nuclease activity (11,23), produced fulllength ssDNA during DNA unwinding but showed no fragment extending from Chi (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…This is in contrast to RecBCD enzyme, which requires a Chi site for significant loading of RecA protein (6), joint molecule formation (7), and recombination (18). The constitutive loading of RecA protein by RecBC enzyme suggested that RecD inhibits RecA loading by the holoenzyme until an interaction with Chi (16).…”

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confidence: 99%

“…Genetic analysis of recD null mutants and examination of purified enzyme led to the hypothesis that RecD acts as an inhibitor of recombination (11) until it is modified or ejected after a RecBCD enzyme-Chi interaction (12,16). This model is supported by the fact that recD mutants are recombination-proficient (11,23,29) and hyperre-combination-proficient in the absence of Chi sites (11), and they cluster recombination events at the ends of DNA in lambda replication-blocked crosses (30).…”

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confidence: 99%

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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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confidence: 71%

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confidence: 79%

Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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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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Stress‐induced mutation via DNA breaks in Escherichia coli: A molecular mechanism with implications for evolution and medicine

et al. 2012

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Evolutionary theory assumed that mutations occur constantly, gradually, and randomly over time. This formulation from the “modern synthesis” of the 1930s was embraced decades before molecular understanding of genes or mutations. Since then, our labs and others have elucidated mutation mechanisms activated by stress responses. Stress-induced mutation mechanisms produce mutations, potentially accelerating evolution, specifically when cells are maladapted to their environment, that is, when they are stressed. The mechanisms of stress-induced mutation that are being revealed experimentally in laboratory settings provide compelling models for mutagenesis that propels pathogen–host adaptation, antibiotic resistance, cancer progression and resistance, and perhaps much of evolution generally. We discuss double-strand-break-dependent stress-induced mutation in Escherichia coli. Recent results illustrate how a stress response activates mutagenesis and demonstrate this mechanism's generality and importance to spontaneous mutation. New data also suggest a possible harmony between previous, apparently opposed, models for the molecular mechanism. They additionally strengthen the case for anti-evolvability therapeutics for infectious disease and cancer.

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The Bacillus subtilis AddAB helicase/nuclease is regulated by its cognate Chi sequence in vitro

Chédin

Ehrlich

Kowalczykowski

2000

Journal of Molecular Biology

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The RecBC enzyme loads RecA protein onto ssDNA asymmetrically and independently of chi , resulting in constitutive recombination activation

Cited by 145 publications

References 62 publications

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

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

Stress‐induced mutation via DNA breaks in Escherichia coli: A molecular mechanism with implications for evolution and medicine

The Bacillus subtilis AddAB helicase/nuclease is regulated by its cognate Chi sequence in vitro

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