recD: the gene for an essential third subunit of exonuclease V.

Amundsen, Susan K.; Taylor, Andrew F.; Chaudhury, Abed; Smith, Gerald R.

doi:10.1073/pnas.83.15.5558

Cited by 254 publications

(211 citation statements)

References 35 publications

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“…The former possibility is consistent with the observation that, although the enzyme appears to be greater than 95% pure on Coomassie-stained gels, the specific activity of our preparation is approximately 11% of that of Amundsen et al (1986), who report the highest specific activity found in the literature (fraction V, 3 X lo5 nuclease units/mg of protein). Our specific activity falls within the range reported by other laboratories (2.1 X lo4 to 2.7 X lo5 nuclease units/mg of protein; Nobrega et al, 1972;Ponticelli et al, 1985;Taylor & Smith, 1985;Hermanns & Wackernagel, 1977;Dykstra et al, 1984).…”

Section: Resultssupporting

confidence: 93%

“…Our value is similar to that of fraction IV of Amundsen et al (1986) and those of Ponticelli et al (1985) and Taylor and Smith (1985). Assuming a molecular weight of 330K for recBCD enzyme, our preparation was calculated to have 5.4 X loio enzyme molecules/unit of nuclease activity or 1.12 x IO4 units/nmol of enzyme.…”

Section: Roman and Kowalczykowskisupporting

confidence: 92%

“…The final enzyme had a specific activity of 3.4 X lo4 nuclease units/mg, only 11% of the value of fraction V of Amundsen et al (1986), who report the highest specific activity achieved to date (3 X lo5 units/mg). Our value is similar to that of fraction IV of Amundsen et al (1986) and those of Ponticelli et al (1985) and Taylor and Smith (1985).…”

Section: Roman and Kowalczykowskimentioning

confidence: 73%

“…Our preparation of recBCD enzyme has a specific activity which is 11% that of Amundsen et al (1986; fraction V) and is calculated to consist of approximately 6% active enzyme. Therefore, the preparation of Amundsen et al (1986) is 6%/11%, or 55%, active. This implies either that their preparation is only half-active or that the functional recBCD helicase unit is a dimer.…”

Section: Resultsmentioning

confidence: 99%

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Characterization of the helicase activity of the Escherichia coli recBCD enzyme using a novel helicase assay

Roman¹,

1989

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W e describe an assay to measure the extent of enzymatic unwinding of D N A by a D N A helicase. This assay takes advantage of the quenching of the intrinsic protein fluorescence of Escherichia coli SSB protein upon binding to ssDNA and is used to characterize the D N A unwinding activity of recBCD enzyme. Unwinding in this assay is dependent on the presence of recBCD enzyme and linear dsDNA, is consistent with the known properties of recBCD enzyme, and closely parallels other methods for measuring recBCD enzyme helicase activity. The effects of varying temperature, substrate concentrations, enzyme concentration, and mono-and divalent salt concentrations on the helicase activity of recBCD enzyme were characterized. when corrected for observed stoichiometry]. With increasing NaCl concentration, k,, peaks a t 100 mM, and the apparent K , value for dsDNA increases by 3-fold a t 200 m M NaC1. In the presence of 5 m M calcium acetate, the apparent K , value is increased by 3-fold, and k,, decreased by 20-30%. W e have also shown that recBCD enzyme molecules are able to catalytically unwind additional dsDNA substrates subsequent to initiation, unwinding, and dissociation from a previous dsDNA molecule. R e c B C D enzyme is a 330-kDa protein consisting of three nonidentical subunits with five different activities. It has DNA-dependent ATPase, single-stranded DNA (ssDNA)' and double-stranded DNA (dsDNA) exonuclease, ssDNA endonuclease, and DNA helicase activities [for reviews, see Telander-Muskavitch and Linn (1981) and Taylor (1988)l. Escherichia coli mutants lacking the recBCD enzyme show a phenotypic reduction in recombination after conjugation or generalized transduction by as much as 3 orders of magnitude and exhibit a reduced ability to recover from DNA damage. Together, recBCD enzyme and recA protein define the RecBCD pathway of recombination, the major pathway used by E. coli. RecBCD enzyme has also been shown to nick dsDNA very specifically at x sites (Taylor et al., 1985), major recombinational uhotspots" [for a review of x sites, see Stahl The nuclease activities of recBCD enzyme are inhibited to varying degrees by SSB protein, which binds to ssDNA and protects it from recBCD enzyme nuclease (Mackay & Linn, 1976), by the presence of calcium ion (Rosamond et al., 1979), and by high levels of ATP. The degradation of dsDNA by recBCD enzyme requires ATP and exhibits an optimum at 20 pM ATP, where the final products of the reaction are single-stranded oligonucleotides three to eight bases long (in the absence of SSB protein and calcium ions) (Eichler & Lehman, 1977;Goldmark & Linn, 1972). The extent of exonuclease activity is inhibited at high (>200 pM) concentrations of ATP while the initial rate of nuclease activity remains unaffected. The products of the reaction under conditions of high ATP concentration are pieces of ssDNA with lengths ranging from 135 to 1400 nucleotides (Mackay & Linn, 1976). This difference in products at low and high ATP concentrations led Mackay and Linn (1976) to hypothesize ...

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Section: Resultssupporting

confidence: 93%

Section: Roman and Kowalczykowskisupporting

confidence: 92%

Section: Roman and Kowalczykowskimentioning

confidence: 73%

Section: Resultsmentioning

confidence: 99%

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Characterization of the helicase activity of the Escherichia coli recBCD enzyme using a novel helicase assay

Roman¹,

1989

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“…recC mutants are recombination deficient, recD mutants are recombination proficient (Chaudhury and Smith, 1984;Amundsen et al, 1986;Thaler et al, 1989) and that the χ stimulation is not detected in recD mutants (Chaudhury and Smith, 1984). The nuclease activity of RecBCD enzyme is undetectable in cell-free extracts prepared from recD mutants (Chaudhury and Smith, 1984).…”

Section: Introductionmentioning

confidence: 98%

DNA replication triggered by double-stranded breaks in E. coli: Dependence on homologous recombination functions

Asai

Bates

Kogoma

1994

Cell

112

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SummaryHomologous recombination-dependent DNA replication (RDR) of a λ cos site-carrying plasmid is demonstrated in E. coli cells when the cells express λ terminase that introduces a double-stranded break into the cos site. RDR occurs in normal wild-type cells if the plasmid also contains the recombination hotspot χ. χ is dispensable when cells are induced for the SOS response or contain a recD mutation. recBC sbcA mutant cells are also capable of RDR induction. A recN mutation greatly reduces RDR in normal cells, but not in SOS-induced cells. RDR proceeds by the θ mode or rolling circle mode of DNA synthesis, yielding covalently closed circular plasmid monomers or linear plasmid multimers, respectively. Previously described inducible stable DNA replication is considered to be a special type of RDR that starts exclusively from specific sites (or/Ms) on the chromosome.

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Homologous recombination-dependent initiation of DNA replication from DNA damage-inducible origins in Escherichia coli.

et al. 1993

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Escherichia coli cells induced for the SOS response express inducible stable DNA replication (iSDR) as an SOS function. Initiation of iSDR is independent of transcription, translation and DnaA protein, which are essential for initiation of DNA replication from oriC. We found that a recA mutant that is defective in recombination but proficient in SOS induction could not elicit iSDR. In contrast, iSDR was enhanced by recD and recJ mutations that inactivate the exonuclease V activity of the RecBCD enzyme and the RecJ exonuclease activity, respectively. A mutation in the ruvC gene that blocks the resolution of recombination intermediates (i.e. Holliday structures) also enhanced iSDR. Furthermore, inhibition of branch migration by recG or ruvAB mutations dramatically increased the iSDR activity. recBC mutants are defective in iSDR induction but the defect was suppressed by a mutation in the sbcA gene. The major product of minichromosomes replicated by iSDR was covalently closed circular monomers. We propose that recombination intermediates (i.e. D‐loop structures) created by the action of RecA recombinase and RecBC(D) helicase play a central role in initiation of iSDR.

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recD: the gene for an essential third subunit of exonuclease V.

Cited by 254 publications

References 35 publications

Characterization of the helicase activity of the Escherichia coli recBCD enzyme using a novel helicase assay

Characterization of the helicase activity of the Escherichia coli recBCD enzyme using a novel helicase assay

DNA replication triggered by double-stranded breaks in E. coli: Dependence on homologous recombination functions

Homologous recombination-dependent initiation of DNA replication from DNA damage-inducible origins in Escherichia coli.

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