Evidence that recBC-dependent degradation of duplex DNA in Escherichia coli recD mutants involves DNA unwinding

Rinken, R; Thomas, Barbara J.; Wackernagel, Wilfried

doi:10.1128/jb.174.16.5424-5429.1992

Cited by 67 publications

(44 citation statements)

References 43 publications

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“…3). This instability of linear DNA in recD mutants is consistent with the high residual levels of linear DNA degradation observed in these mutants and is ascribed to the action of RecBC helicase converting duplex DNA into a substrate for single-strand DNA-specific nucleases (38). Since linearized DNA in a recA strain is stable with time, we believe that a fraction of the cells in a recA culture are partial recBC phenocopies.…”

Section: ϫ8supporting

confidence: 56%

Stability of linear DNA in recA mutant Escherichia coli cells reflects ongoing chromosomal DNA degradation

Kuzminov

Stahl

1997

J Bacteriol

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To study the fate of linear DNA in Escherichia coli cells, we linearized plasmid DNA at a specific site in vivo and monitored its behavior in recA mutant cells deficient in recombinational repair. Earlier, we had found that in wild-type (WT) cells linearized DNA is degraded to completion by RecBCD nuclease. We had also found that in WT cells sites on linear DNA inhibit RecBCD degradation by turning off its nucleolytic activities. Now we report that sites do not work in the absence of the RecA protein, suggesting that RecA is required in vivo to turn off the degradative activities of the RecBCD enzyme. We also report that the degradation of linearized plasmid DNA, even devoid of sites, is never complete in recA cells. Investigation of this linear DNA stability indicates that a fraction of recA cells are recBC phenocopies due to ongoing chromosomal DNA degradation, which titrates RecBCD nuclease. A possible role for RecBCD-promoted DNA degradation in controlling chromosomal DNA replication in E. coli is discussed.ExoV of Escherichia coli, the most powerful exonuclease known, is a heterotrimer of RecB, RecC, and RecD subunits (49,51). It attacks only DNA with free ends and combines helicase activity with duplex DNA-specific as well as singlestranded DNA-specific nuclease activities. In vitro, the enzyme proceeds along the DNA with a speed of up to 1 kb/s (39), unwinding 30 kbp per binding event (40). In vivo, RecBCD not only degrades unprotected linear DNA but also cooperates with the RecA protein to carry out recombinational repair of double-strand breaks and disintegrated replication forks (26,27). The transition from a destructive to a repair enzyme is facilitated by encounters with signal sequences in the DNA being degraded. These asymmetric octanucleotide sequences are called sites; they occur about once per 4 kb of the E. coli genome (34).The complex behavior of RecBCD in the vicinity of a site is now understood in broad outline (34). To "see" a site, the enzyme must approach it from a particular direction. When RecBCD sees , its DNA-degradative activities are modified so as to assist in RecA-promoted recombinational repair; this modification persists as long as the frayed end is searching for homologous intact DNA with which to pair (35). The modification of RecBCD involves some kind of inactivation of the RecD subunit (19,25,33), an idea originally put forward to explain the observation that the RecBC(D Ϫ ) mutant enzyme behaves as though it were permanently activated by sites (52). recD mutant cells lack ExoV but are proficient in recombinational repair and have normal viability (1). In contrast, recB or recC mutants, also ExoV Ϫ , are deficient in recombinational repair of double-strand ends (41) and have poor viability (7). recA mutants, although most severely deficient in recombinational repair (41), are more viable than recBC mutants (7), a phenomenon which has not been explained.To study metabolism of linear DNA in vivo, we have constructed several cosmids-plasmids bearing the cos site of phage lambda...

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Section: ϫ8supporting

confidence: 56%

Stability of linear DNA in recA mutant Escherichia coli cells reflects ongoing chromosomal DNA degradation

Kuzminov

Stahl

1997

J Bacteriol

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“…Note that nuclease inactivation may even be underestimated in this assay, as unwinding activity alone may have a modest inhibitory effect on T4g2 multiplication (40). This result shows that the RexB nuclease motif DYK is a functionally active component of the exo/hel enzyme.…”

Section: Resultsmentioning

confidence: 66%

In Vivo Evidence for Two Active Nuclease Motifs in the Double-Strand Break Repair Enzyme RexAB of Lactococcus lactis

et al. 2001

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In bacteria, double-strand DNA break (DSB) repair involves an exonuclease/helicase (exo/hel) and a short regulatory DNA sequence (Chi) that attenuates exonuclease activity and stimulates DNA repair. Despite their key role in cell survival, these DSB repair components show surprisingly little conservation. The best-studied exo/hel, RecBCD of Escherichia coli, is composed of three subunits. In contrast, RexAB of Lactococcus lactis and exo/hel enzymes of other low-guanine-plus-cytosine branch gram-positive bacteria contain two subunits. We report that RexAB functions via a novel mechanism compared to that of the RecBCD model. Two potential nuclease motifs are present in RexAB compared with a single nuclease in RecBCD. Site-specific mutagenesis of the RexA nuclease motif abolished all nuclease activity. In contrast, the RexB nuclease motif mutants displayed strongly reduced nuclease activity but maintained Chi recognition and had a Chi-stimulated hyperrecombination phenotype. The distinct phenotypes resulting from RexA or RexB nuclease inactivation lead us to suggest that each of the identified active nuclease sites in RexAB is involved in the degradation of one DNA strand. In RecBCD, the single RecB nuclease degrades both DNA strands and is presumably positioned by RecD. The presence of two nucleases would suggest that this RecD function is dispensable in RexAB.In bacteria, double-stranded DNA breaks (DSB) are frequent events that may be provoked, for example, by pauses in the replication fork (36, 43). Such genomic disruptions are lethal in the absence of DNA repair. In Escherichia coli DSB repair requires the activity of a large enzyme complex, known as RecBCD, that has ATP-dependent helicase and exonuclease activities (see reference 32 for a review). The enzyme degrades both strands, starting from the DNA break until it reaches an octanucleotide sequence, known as Chi, that attenuates degradation and stimulates recombination (44,46). The enzyme exhibits helicase activity and residual exonuclease activity with an altered polarity after Chi (4, 16); the remaining activity provides a single-stranded DNA substrate for recombination enzymes to mediate repair.Organization of the three-subunit exonuclease/helicase (exo/hel) RecBCD. Structure-functional studies of RecBCD have revealed some of the roles of each subunit. RecB seems to possess two key activities of the enzyme. The N-terminal 929 amino acids (out of 1,180 total) have confirmed ATPase and helicase activities (13,54

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“…Thus, the consequence of RecBC enzyme helicase and RecJ protein nuclease action on a dsDNA end would be to produce a 3 0 -ssDNA overhang. In fact, the recBC-, recJ-dependent degradation of ssDNA occurs in vivo (Rinken et al 1992).…”

Section: Discussionmentioning

confidence: 99%

Chi‐activated RecBCD enzyme possesses 5′→3′ nucleolytic activity, but RecBC enzyme does not: evidence suggesting that the alteration induced by Chi is not simply ejection of the RecD subunit

1997

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Background: Homologous recombination in Escherichia coli is initiated by the RecBCD enzyme, and is stimulated by DNA elements known as Chi (x) sites. The RecBCD enzyme is both a helicase and a nuclease. Recognition of x causes both attenuation of the 3 0 →5 0 exonuclease activity of the RecBCD enzyme, and activation of an exonuclease activity with 5 0 →3 0 polarity, while leaving the helicase activity unaffected. A variety of evidence suggests that x-recognition by RecBCD enzyme is accompanied by ejection of the RecD subunit.

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Evidence that recBC-dependent degradation of duplex DNA in Escherichia coli recD mutants involves DNA unwinding

Cited by 67 publications

References 43 publications

Stability of linear DNA in recA mutant Escherichia coli cells reflects ongoing chromosomal DNA degradation

Stability of linear DNA in recA mutant Escherichia coli cells reflects ongoing chromosomal DNA degradation

In Vivo Evidence for Two Active Nuclease Motifs in the Double-Strand Break Repair Enzyme RexAB of Lactococcus lactis

Chi‐activated RecBCD enzyme possesses 5′→3′ nucleolytic activity, but RecBC enzyme does not: evidence suggesting that the alteration induced by Chi is not simply ejection of the RecD subunit

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