Interaction of RecBCD enzyme with DNA damaged by gamma radiation

Brčić-Kostić, Krunoslav; Salaj-Šmic, Erika; Maršić, Nataša; Kajić, S; Stojiljković, Igor; Trgovčević, Željko

doi:10.1007/bf00282458

Cited by 18 publications

(14 citation statements)

References 41 publications

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“…The nature of this titration is revealed by the fact that recA mutant cells degrade their chromosomal DNA if it is damaged (14), the degradation being carried out by RecBCD nuclease (56). During rapid chromosomal degradation caused by fragmentation with gamma radiation or bleomycin, E. coli cells become ExoV Ϫ mutant phenocopies in that they plate T4 2 mutant phage (5,25), demonstrating that RecBCD nuclease is titrated during chromosomal degradation. A fraction of the cells in a growing recA culture could also be ExoV Ϫ due to ongoing DNA degradation of the spontaneously damaged chromosome, which would result in increased survival of linearized cosmid DNA.…”

Section: Discussionmentioning

confidence: 99%

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: Discussionmentioning

confidence: 99%

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

Kuzminov

Stahl

1997

J Bacteriol

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“…Extensive chromosomal breakage induced by DNAdamaging agents, for example, leads to the inhibition of RecBCD in vivo (42,150,170,232,298). Further studies with plasmids led to the conclusion that the interaction with Chi sequences produced inactivation and that a RecA mutation affected the phenomenon (169,170).…”

Section: Host Dna Degradationmentioning

confidence: 99%

RecBCD Enzyme and the Repair of Double-Stranded DNA Breaks

Dillingham

Kowalczykowski

2008

Microbiol Mol Biol Rev

503

599

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SUMMARY The RecBCD enzyme of Escherichia coli is a helicase-nuclease that initiates the repair of double-stranded DNA breaks by homologous recombination. It also degrades linear double-stranded DNA, protecting the bacteria from phages and extraneous chromosomal DNA. The RecBCD enzyme is, however, regulated by a cis-acting DNA sequence known as Chi (crossover hotspot instigator) that activates its recombination-promoting functions. Interaction with Chi causes an attenuation of the RecBCD enzyme's vigorous nuclease activity, switches the polarity of the attenuated nuclease activity to the 5′ strand, changes the operation of its motor subunits, and instructs the enzyme to begin loading the RecA protein onto the resultant Chi-containing single-stranded DNA. This enzyme is a prototypical example of a molecular machine: the protein architecture incorporates several autonomous functional domains that interact with each other to produce a complex, sequence-regulated, DNA-processing machine. In this review, we discuss the biochemical mechanism of the RecBCD enzyme with particular emphasis on new developments relating to the enzyme's structure and DNA translocation mechanism.

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“…While ExoV is the strongest duplex DNA exonuclease in E. coli active in the degradation of restricted foreign DNA (64) or unprotected infecting phage genomes like that of T4 2 Ϫ (55), its activity rapidly disappears when the enzyme interacts in vivo with linear DNA containing Chi sequences. Silencing occurs during rolling-circle replication of Chi-containing plasmids (20) or after restriction of phage DNA with Chi sites (26; B. Thoms and W. Wackernagel, unpublished data), fragmentation of the E. coli genome by gamma-irradiation of cells (6), or treatment with bleomycin (30). It was assumed that the blunt duplex DNA ends produced by these treatments allowed RecBCD to attack the DNA and rapidly encounter Chi sites, where the enzyme is silenced.…”

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Effects of Single-Strand DNases ExoI, RecJ, ExoVII, and SbcCD on Homologous Recombination of recBCD ⁺ Strains of Escherichia coli and Roles of SbcB15 and XonA2 ExoI Mutant Enzymes

2008

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To assess the contributions of single-strand DNases (ssDNases) to recombination in a recBCD ؉ background, we studied 31 strains with all combinations of null alleles of exonuclease I (⌬xon), exonuclease VII (xseA), RecJ DNase (recJ), and SbcCD DNase (sbcCD) and exonuclease I mutant alleles xonA2 and sbcB15. The xse recJ sbcCD ⌬xon and xse recJ sbcCD sbcB15 quadruple mutants were cold sensitive, while the quadruple mutant with xonA2 was not. UV sensitivity increased with ssDNase deficiencies. Most triple and quadruple mutants were highly sensitive. The absence of ssDNases hardly affected P1 transductional recombinant formation, and conjugational recombinant production was decreased (as much as 94%) in several cases. Strains with sbcB15 were generally like the wild type. We determined that the sbcB15 mutation caused an A183V exchange in exonuclease motif III and identified xonA2 as a stop codon eliminating the terminal 8 amino acids. Purified enzymes had 1.6% (SbcB15) and 0.9% (XonA2) of the specific activity of wild-type Xon (Xon ؉ ), respectively, with altered activity profiles. In gel shift assays, SbcB15 associated relatively stably with 3 DNA overhangs, giving protection against Xon ؉ . In addition to their postsynaptic roles in the RecBCD pathway, exonuclease I and RecJ are proposed to have presynaptic roles of DNA end blunting. Blunting may be specifically required during conjugation to make DNAs with overhangs RecBCD targets for initiation of recombination. Evidence is provided that SbcB15 protein, known to activate the RecF pathway in recBC strains, contributes independently of RecF to recombination in recBCD ؉ cells. DNA end binding by SbcB15 can also explain other specific phenotypes of strains with sbcB15.The heterotrimeric RecBCD enzyme (encoded by the recB, recC, and recD genes) is a central component of the main pathway of genetic recombination and recombinational DNA repair of Escherichia coli (the RecBCD pathway) and functions in the initiation of these processes (31,34,37). The enzyme (also termed exonuclease V [ExoV]), with its DNase and helicase activities, processively degrades duplex DNA from an end until it reaches an octanucleotide termed Chi, which is present in the E. coli genome once per 5,000 nucleotides on average. Upon contact with Chi, the duplex DNA degradation activity of the enzyme is attenuated and switched to produce a 3Ј single-stranded (ss) DNA end on which it loads RecA protein, making a nucleoprotein filament ready to initiate recombination. recB or recC null mutations drastically reduce homologous recombination, increase the sensitivity of cells to DNA-damaging agents, and impair cell viability (34). In several studies extragenic suppressors of the severe effects of recBC mutations have been isolated and characterized. One group, termed sbcA mutations, was found to express recombination genes of the cryptic Rac prophage (43,68). The other group of mutations was located in the gene for ExoI and affected ExoI activity (32,33). ExoI is a 3Ј-specific processive exonuclease for ...

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Interaction of RecBCD enzyme with DNA damaged by gamma radiation

Cited by 18 publications

References 41 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

RecBCD Enzyme and the Repair of Double-Stranded DNA Breaks

Effects of Single-Strand DNases ExoI, RecJ, ExoVII, and SbcCD on Homologous Recombination of recBCD ⁺ Strains of Escherichia coli and Roles of SbcB15 and XonA2 ExoI Mutant Enzymes

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Interaction of RecBCD enzyme with DNA damaged by gamma radiation

Cited by 18 publications

References 41 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

RecBCD Enzyme and the Repair of Double-Stranded DNA Breaks

Effects of Single-Strand DNases ExoI, RecJ, ExoVII, and SbcCD on Homologous Recombination of recBCD + Strains of Escherichia coli and Roles of SbcB15 and XonA2 ExoI Mutant Enzymes

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Effects of Single-Strand DNases ExoI, RecJ, ExoVII, and SbcCD on Homologous Recombination of recBCD ⁺ Strains of Escherichia coli and Roles of SbcB15 and XonA2 ExoI Mutant Enzymes