recBC DNase Research Articles

Factor which affects the mode of genetic recombination in E. Coli.

THE normal recombination process in Escherichia coli requires a pathway involving a functional recB and recC gene product (henceforth designated the RecBC pathway)1. The recB and recC genes map as closely linked cistrons near thy on the E. coli chromosome map (54 min)2,3. ATP-dependent DNase (recBC DNase) has been identified as the product encoded by these genes4–6. Another gene essential for recombination proficiency, recA1, is located between cysC and pheA (51 min)8. Genetic transformation studies9–12 showed that the most efficiently transformable E. coli strain (genotype: recB− recC− sbcB−) lacks the DNase but retains recombination proficiency because of the sbcB− allele which acts as an indirect suppressor of the recB− recC− mutations13. This strain uses a recombination pathway (designated as the RecF pathway)1 which does not involve the ATP-dependent DNase but consists of products of several recombination genes13. Restoration of the ATP-dependent DNase to this strain by introduction of the recB+ recC+ alleles reduced the transformation frequency by a factor of approximately five9. In vitro studies demonstrated that this DNase degrades linear DNA molecules extensively, while it has no effect on circular DNA4. The adverse effect of the DNase on transformation may therefore be due to destruction of linear donor DNA molecules before they reach the recombination machinery. These results raise the possibility that in normal cells the DNase affects the mode of recombination by selecting or destroying DNA molecules of a particular structure.

The degradation of duplex DNA by the recBC DNase of Escherichia coli.

The catalytic reactions of the recBC enzyme of Escherichia coli with various substrates are reviewed, and a model for the sequence of events in the degradation of duplex DNA is presented. The potential of the enzyme to take part in genetic recombination and repair is discussed.

The recBC deoxyribonuclease of Escherichia coli: isolation and characterization of the subunit proteins and reconstitution of the enzyme.

After dissociation of the E. coli recBc DNase (ATP-dependent DNase) with concentrated NaCl, two subunit proteins were isolated by ion exchange chromatography. Combination and subsequent incubation of the subunits resulted in the appearance of the original DNase. The subunit proteins, designated alpha and beta, have s(20,omega) of 4.1 S and 8.1 S, respectively. The alpha subunit possesses neither the ATP-dependent Dnase nor the DNA-dependent ATPase of the original enzyme. The beta subunit contains a low level of both enzymatic activities in a ratio markedly different from that of the original enzyme. The beta subunit complemented extracts from both recB and recC mutant strains to produce recBC DNase, while the alpha subunit did not complement either extract. These results suggest that recB and recC genes are both required for the production of beta subunit and that the recBC DNase molecule contains a protein component (alpha) that is not determined by either the recB or the recC gene.

Inhibition of exonuclease V after infection of [formula omitted] by bacteriophage T7

Exonuclease V ( recBC DNase) is inactivated in E. coli between 4 and 7 min after infection by T7. This process requires protein sythesis. The inactivation does not occur when T7 is deficient for its RNA polymerase and thus does not express the genes involved in DNA replication and phage maturation. Some implications of this new function of T7 are discussed with respect to the processes of infection and DNA replication.

The Mechanism of Degradation of Duplex Deoxyribonucleic Acid by the recBC Enzyme of Escherichia coli K-12

Abstract Reaction intermediates formed during the degradation of duplex DNA by the recBC DNase have been characterized by sedimentation velocity, isopyknic centrifugation, electron microscopy, and susceptibility to various deoxyribonucleases which are specific for single-stranded DNA. It has been concluded that the intermediates are of two classes: (a) duplex molecules with single-stranded tails of several thousand nucleotides at the 3' and the 5' termini and (b) singlestranded fragments several hundred nucleotides in length. To explain the presence of these intermediates, even in synchronized reactions with DNA molecules in molar excess over enzyme, a reaction scheme for the degradation of duplex DNA is proposed. According to this scheme, the DNase unwinds the strands of the duplex from an end and clips off fragments several hundred nucleotides long from one strand, while remaining bound to the terminus of the undegraded strand. When a single-stranded tail of up to several thousand nucleotides is formed in this way, the enzyme switches strands and degrades the tail from its terminus, yielding a shortened, wholly duplex molecule. The cycle is then repeated until the molecule is entirely digested. The scheme recognizes the processive nature of the degradation of duplex DNA by this enzyme and hypothesizes that the ATP hydrolysis which accompanies DNA degradation is required for the unwinding reaction.

Purification and Properties of the γ-Protein Specified by Bacteriophage λ: An Inhibitor of the Host RecBC Recombination Enzyme

Previous experiments have indicated that the gam gene of bacteriophage lambda is responsible for an inhibition of the RecBC DNase-an enzyme that is essential for the major host pathway of genetic recombination. We report here experiments that define the inhibitor as the protein product of the gam gene ("gamma-protein") and that characterize the inhibition reaction with highly purified preparations of gamma-protein and RecBC DNase. Genetic characterization was performed with partially purified fractions prepared from cells infected with various lambda mutants. An activity that inhibits RecBC DNase was absent in extracts prepared after infection by phage that carry nonsense or deletion mutations in the gam gene; this activity was highly thermolabile in an extract prepared after infection by phage that carry a temperature-sensitive mutation in the gam gene. For biochemical characterization, the gamma-protein has been purified more than 800-fold. This highly purified preparation inhibited all of the known catalytic activities associated with the RecBC enzyme, but exhibited no detectable DNase or ATPase activities by itself. These findings are discussed in terms of their implications for regulation of genetic recombination and bacteriophage lambda development.

The recBC Deoxyribonuclease of Escherichia coli K-12: SUBSTRATE SPECIFICITY AND REACTION INTERMEDIATES

Abstract The behavior of the recBC DNase of Escherichia coli with a variety of substrates has been investigated. Duplex circular DNA is resistant to digestion, is not a cofactor for the DNA-dependent ATPase, and does not effectively compete for enzyme with substrate DNAs. Treatment of duplex circles with ultraviolet light or x-rays does not render them susceptible to the enzyme nor does nicking with pancreatic DNase. In the latter case the nicks are neither sealed nor translocated. Removal of the phosphomonoester group from a nick has no effect, but localized denaturation or removal of approximately five nucleotides at a nick renders duplex circles susceptible to digestion. The enzyme appears to digest linear DNA by a processive mechanism in which the rate is limited by the initiation of digestion of a molecule. It shows no apparent polarity of degradation, nor does it appear to be affected by the presence or absence of terminal phosphate groups. The original terminal nucleotides are dispersed among all classes of the products in a limit digest, so they do not constitute a special type of product. Linear DNA treated with ultraviolet light or x-rays is degraded by the enzyme, but RNA-DNA hybrid is resistant. The latter substrate can serve as cofactor for the DNA-dependent ATPase, however, effectively uncoupling the two activities. At ATP concentrations of 1 mm or greater the enzyme generates early in the reaction large, sedimentable DNA fragments, as well as nonsedimentable products. The large fragments are primarily duplex, but contain single-stranded regions at 5' termini and probably at 3' termini at roughly equal frequency.

Transfection by half molecules and inverted molecules of λ DNA: Requirement for exo and β-promoted recombination

A rec + strain of E. coli was infected less efficiently by half molecules and inverted molecules of λ DNA than were recombination deficient strains that lack the recBC DNase. In recB spheroplasts, transfection by half molecules or inverted molecules depends on the action of both of the principal genes of the phage λ system for recombination, namely, the genes for λ exonuclease and β protein. Partial digestion of the ends of inverted molecules by λ exonuclease in vitro bypasses the requirement for the action of either the exo or β gene. Thus, in recB, transfection by broken molecules of DNA with either double-stranded or single-stranded ends does not discriminate between the roles of exonuclease and β protein in genetic recombination. When the recB defect is suppressed by either sbcA or sbcB mutations, neither half molecules nor inverted molecules require λ exonuclease or β protein for infectivity. In recB, the dependence of half molecules and inverted molecules on the genes for exonuclease and β protein for infectivity shows that these genes are active in DNA molecules that are not circular. Furthermore, since the product of the N gene is required for expression of the genes for exonuclease and β protein, circularization of the DNA is probably not a prerequisite for the transcription of N.

Genetic transformation in [formula omitted]: The inhibitory role of the [formula omitted] DNase

Genetic transformation of E. coli for various chromosomal markers was accomplished by (i) using recipient cells that lack the recBC DNase but were recombination proficient due to sbcA or sbcB mutations and (ii) treating the recipient cells with CaCl 2 at a concentration that facilitates transfection by λ DNA. Cotransformation of three markers ( thr + ara +. leu + ) was found to depend on the molecular weight of the transforming DNA.

Purification and Properties of the recBC DNase of Escherichia coli K-12

Abstract The deoxyribonuclease controlled by the recB and recC loci of Escherichia coli K-12 has been purified to near homogeneity. It is composed of two nonidentical polypeptide chains with a combined molecular weight of approximately 270,000. In the presence of ATP the enzyme hydrolyzes linear duplex or single stranded DNA in an exonucleolytic manner to 5'-phosphoryl-terminated oligonucleotides of an average length of 4.5. The enzyme can also cleave single stranded DNA endonucleolytically, being stimulated 7-fold by ATP in this reaction. However, no nuclease activity is observed on closed circular duplex DNA. In the course of exonuclease digestion more than 20 ATP molecules are hydrolyzed to ADP and inorganic phosphate for each DNA phosphodiester bond broken. There is no ATP hydrolysis in the absence of degradable DNA.