Pyrimidine dimer excision in Escherichia coli strains deficient in exonucleases V and VII and in the 5'' leads to 3'' exonuclease of DNA polymerase I.

An isogenic series of Escherichia coli strains deficient in various combinations of three 5' leads to 3' exonucleases (exonuclease V, exonuclease VII, and the 5' leads to 3' exonuclease of DNA polymerase I) was constructed and examined for the ability to excise pyrimidine dimers after UV irradiation. Although the recB and recC mutations (deficient in exonuclease V) proved to be incompatible with the polA(Ex) mutation (deficient in the 5' leads to 3' exonuclease of DNA polymerase I), it was possible to reduce the level of the recB,C exonuclease by the use of temperature-sensitive recB270 recC271 mutants. It was found that, by employing strains deficient in exonuclease V, postirradiation DNA degradation could be reduced and dimer excision measurements could be facilitated. Mutants deficient in exonuclease V were found to excise dimers at a rate comparable to that of the wild type. Mutants deficient in exonuclease V and the 5' leads to 3' exonuclease of DNA polymerase I are slightly slower than the wild type at removing dimers accumulated after doses in excess of 40 J/m2. However, although strains with reduced levels of exonuclease VII excised dimers at the same rate as the wild type, the addition of an exonuclease VII deficiency to a strain with reduced levels of exonuclease V and the 5' leads to 3' exonuclease of DNA polymerase I caused a marked decrease in the rate and extent of dimer excision. These observations support previous indications that the 5' leads to 3' exonuclease of DNA polymerase I is important in dimer removal and also suggest a role for exonuclease VII in the excision repair process.     

On the role of ATP in phosphodiester bond hydrolysis catalyzed by the recBC deoxyribonuclease of Escherichia coli.

The deoxyribonuclease specified by the recB and recC genes of Escherichia coli (recBC DNase; exonuclease V) has been purified to near homogeneity by a new procedure. Although hydrolysis of even a single nucleotide from a duplex DNA molecule by the pure enzyme is absolutely dependent upon ATP, the extent of phosphodiester hydrolysis is strongly inhibited by ATP concentrations of 0.2 mm or greater, and the initial rate is unaffected. Under these conditions, the extent of DNA hydrolysis is proportional to enzyme concentration. In contrast, neither the rate nor the extent of hydrolysis of single-stranded DNA nor ATP is affected by high concentrations of ATP. The amount of large single-stranded polynucleotide generated by the action of the recBC DNase increases as the ATP concentration increases and, at 0.5 mM ATP, becomes equivalent to the amount of acid-soluble nucleotide formed. These findings suggest that high intracellular concentrations of ATP affect the mechanism of the recBC DNase so as to limit the extent of hydrolysis of duplex DNA, while at the same time favoring the formation of single-stranded regions within the duplex. Such regions may be essential intermediates in the recombination process.     

Properties of the recB and recC gene products of Escherichia coli

The properties of the recB and recC gene products of Escherichia coli were studied using recB and recC gene-inserted plasmids. recB mutants and recC mutants lacked ATP-dependent DNase (recBC enzyme) but showed apparent recovery of enzyme activity on introduction of plasmids carrying the recB and recC gene, respectively. The ATP-dependent DNase was also constructed in vitro by mixing the recB and recC gene products encoded by the plasmids with the corresponding gene. Specific labeling of plasmid-encoded proteins by the maxicell method showed that the recB and recC gene products were 135,000 and 125,000 dalton proteins, respectively. These results suggest that the recB and recC genes are the structural genes of the beta and alpha subunits, respectively, of the recBC enzyme. A gene that encodes a protein of about 100,000 daltons was found to be located between the recB and recC genes. But the product of this gene was shown not to be included in the recBC enzyme.     

RecBC enzyme activity is required for far-UV induced respiration shutoff in Escherichia coli K12

Shutoff of respiration is one of a number of recA+ lexA+ dependent (SOS) responses caused by far ultraviolet (245 nm) radiation (UV) damage of DNA in Escherichia coli cells. Thus far no rec/lex response has been shown to require the recB recC gene product, the RecBC enzyme. We report in this paper that UV-induced respiration shutoff did not occur in either of these radiation-sensitive derivatives of K12 strain AB1157 nor in the recB recC double mutant. The sbcB gene product is exonuclease I and it has been reported that the triple mutant strain recB recC sbcB has near normal recombination efficiency and resistance to UV. The sbcB strain shut off its respiration after UV but the triple mutant did not show UV-induced respiration shutoff; the shutoff and death responses were uncoupled. We concluded that respiration shutoff requires RecBC enzyme activity. The RecBC enzyme has ATP-dependent double-strand exonuclease activity, helicase activity and several other activities. We tested a recBC+ (double dagger) mutant strain (recC 1010) that had normal recombination efficiency and resistance to UV but which possessed no ATP-dependent double-strand exonuclease activity. This strain did not shut off its respiration. The presence or absence of other RecBC enzyme activities in this mutant is not known. These results support the hypothesis that ATP-dependent double-strand exonuclease activity is necessary for UV-induced respiration shutoff.     

Escherichia coli recBC deletion mutants.

Mutants of Escherichia coli with deletions of the recB and recC genes were obtained by two methods using transposable DNA elements. The phenotypes of these mutants are similar to those of mutants with recBC point mutations. These results indicate that the RecBC gene products, exonuclease V, is not essential for the growth of E. coli but is important for DNA repair and recombination.     

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

Infection of Escherichia coli with phage T4 gene 2am was used to transport 3H-labeled linear duplex DNA into cells to follow its degradation in relation to the cellular genotype. In wild-type cells, 49% of the DNA was made acid soluble within 60 min; in recB or recC cells, only about 5% of the DNA was made acid soluble. Remarkably, in recD cells about 25% of the DNA was rendered acid soluble. The DNA degradation in recD cells depended on intact recB and recC genes. The degradation in recD cells was largely decreased by mutations in recJ (which eliminates the 5' single-strand-specific exonuclease coded by this gene) or xonA (which abolishes the 3' single-strand-specific exonuclease I). In a recD recJ xonA triple mutant, the degradation of linear duplex DNA was roughly at the level of a recB mutant. Results similar to those with the set of recD strains were also obtained with a recC++ mutant (in which the RecD protein is intact but does not function) and its recJ, xonA, and recJ xonA derivatives. The observations provide evidence for a recBC-dependent DNA-unwinding activity that renders unwound DNA susceptible to exonucleolytic degradation. It is proposed that the DNA-unwinding activity causes the efficient recombination, DNA repair, and SOS induction (after application of nalidixic acid) in recD mutants. The RecBC helicase indirectly detected here may have a central function in Chi-dependent recombination and in the recombinational repair of double-strand breaks by the RecBCD pathway.     

In Vivo Studies of Temperature-Sensitive recB and recC Mutants

Some in vivo properties of Escherichia coli K-12 strains carrying recB270 (formerly recBts1) and recC271 (formerly recCts1) mutations have been determined. Single recB270 and recC271 mutants appear normal at 30 C with regard to ultraviolet and mitomycin C sensitivity, recombination proficiency, and viability. At 43 C these strains become sensitive to ultraviolet and mitomycin C, while showing only a slight decrease in recombination proficiency. The viable titers of the single mutants are somewhat reduced at 43 C. Double mutant strains carrying polA1 and recB270 or recC271 are inviable at 43 C. The double mutant strain (recB270 recC271) is sensitive to both UV and mitomycin C at 30 C, but shows only slightly reduced recombination proficiency. At 43 C the strain resembles absolute recB and recC mutants in all respects. In addition, the double mutant strain exhibits a temperature-induced drop in viable titer. The triple mutant polA1 recB270 recC271 is viable at 30 C. Two hypotheses are advanced to explain these results.     

Genetic analysis of double-strand break repair in Escherichia coli.

We had reported that a double-strand gap (ca. 300 bp long) in a duplex DNA is repaired through gene conversion copying a homologous duplex in a recB21 recC22 sbcA23 strain of Escherichia coli, as predicted on the basis of the double-strand break repair models. We have now examined various mutants for this repair capacity. (i) The recE159 mutation abolishes the reaction in the recB21C22 sbcA23 background. This result is consistent with the hypothesis that exonuclease VIII exposes a 3'-ended single strand from a double-strand break. (ii) Two recA alleles, including a complete deletion, fail to block the repair in this recBC sbcA background. (iii) Mutations in two more SOS-inducible genes, recN and recQ, do not decrease the repair. In addition, a lexA (Ind-) mutation, which blocks SOS induction, does not block the reaction. (iv) The recJ, recF, recO, and recR gene functions are nonessential in this background. (v) The RecBCD enzyme does not abolish the gap repair. We then examined genetic backgrounds other than recBC sbcA, in which the RecE pathway is not active. We failed to detect the double-strand gap repair in a rec+, a recA1, or a recB21 C22 strain, nor did we find the gap repair activity in a recD mutant or in a recB21 C22 sbcB15 sbcC201 mutant. We also failed to detect conservative repair of a simple double-strand break, which was made by restriction cleavage of an inserted linker oligonucleotide, in these backgrounds. We conclude that the RecBCD, RecBCD-, and RecF pathways cannot promote conservative double-strand break repair as the RecE and lambda Red pathways can.     

Physical and biochemical analysis of the cloned recB and recC genes of Escherichia coli K-12.

A 19-kilobase BamHI fragment encoding the recB (exonuclease V), recC (exonuclease V), ptr (protease III), thyA, and argA genes of Escherichia coli K-12 was cloned into a multicopy plasmid (pCDK3). In E. coli maxicells, the plasmid specified the synthesis of seven polypeptides of 140,000 (recC), 128,000 (recB), 110,000 (ptr), 53,000 (argA), 50,000, 33,000 (thyA), and 22,000 Mr, as well as beta-lactamase and chloramphenicol acetyltransferase. From analysis of subclones and Tn1000 insertions, it appears that the 110,000- and 50,000-Mr proteins originated from the ptr DNA coding sequence which is located between the recB and recC genes. Although recC, ptr, and recB were physically closely linked and transcribed in the same direction, they do not appear to constitute an operon. Cells carrying pCDK3 contained a 30- to 50-fold increase in exonuclease V activity, without affecting cell viability.     

Escherichia coli recA protein protects single-stranded DNA or gapped duplex DNA from degradation by RecBC DNase

RecA- mutants of Escherichia coli extensively degrade their DNA following UV irradiation. Most of this degradation is due to the recBC DNase, which suggests that the recA gene is involved in the control of recBC DNase in vivo. We have shown that purified recA protein inhibits the endonuclease and exonuclease activities of recBC DNase on single-stranded DNA. The extent of inhibition is dependent on the relative concentration of recA protein, recBC DNase, and the DNA substrate; inhibition is greatest when the concentrations of DNA and recBC DNase are low and the concentrations of recA protein is high. At fixed concentrations of recA protein and recBC DNase, inhibition is eliminated at high concentrations of DNA. In the presence of adenosine 5'-O-(3-thiotriphosphate), an ATP analog which stabilizes the binding of recA protein to both single- and double-stranded DNA, recA protein is a more potent inhibitor of the nuclease activities on single-stranded DNA and is a weak inhibitor of the exonuclease activity on double-stranded DNA. Inhibition of the latter is enhanced by oligodeoxynucleotides, which stimulate the binding of recA protein to double-stranded DNA. In the presence of adenosine 5'-O-(3-thiotriphosphate), recA protein also inhibits the action of exonuclease I on single-stranded DNA and of lambda exonuclease on double-stranded DNA. These observations are most consistent with the idea that recA protein protects DNA from recBC DNase by binding to DNA. RecA protein also blocks the endonucleolytic cleavage of gapped circular DNA by recBC DNase. Since both recA protein and recBC DNase have the ability under certain conditions to unwind duplex DNA and to displace strands, we looked for evidence that their combined action would enlarge gaps but found no extensive enlargement. D-loops, a putative intermediate in genetic recombination, are effectively protected against the action of recBC DNase by the E. coli single strand binding protein and by recA protein in the presence of adenosine 5'-O-(3-thiotriphosphate).     

Cloning of the recB, recC, and recD genes from Proteus mirabilis in Escherichia coli: in vivo formation of active hybrid enzymes.

We cloned chromosomal DNA fragments from Proteus mirabilis which complement recBCD deletion mutants of Escherichia coli by restoring (i) recombination proficiency in conjugation, (ii) normal resistance to UV irradiation, and (iii) ATP-dependent exonuclease activity for duplex DNA. The data indicate that the order of the genes thyA, recC, recB, recD, and argA is similar in both P. mirabilis and E. coli. Hybrid enzymes formed in vivo were active in repair and recombination.     

Physical and biochemical characterization of cloned sbcB and xonA mutations from Escherichia coli K-12.

In Escherichia coli K-12, sbcB/xonA is the structural gene for exonuclease I, an enzyme that hydrolyzes single-stranded DNA to mononucleotides in the 3'-to-5' direction. This enzyme has been implicated in the DNA repair and recombination pathways mediated by the recB and recC gene products (exonuclease V). We have cloned several sbcB/xonA mutant alleles in bacterial plasmids and have partially characterized the cloned genes and their protein products. Two of the mutations (xonA2 and xonA6) retain no detectable exonucleolytic activity on single-stranded DNA. The xonA6 allele was shown to harbor an insertion of an IS30-related genetic element near the 3' end of the gene. Two other mutations, sbcB15 and xonA8, exhibited significantly reduced levels of exonuclease I activity as compared to the cloned wild-type gene. A correlation was observed between levels of exonuclease I activity and the ability of the sbcB/xonA mutations to suppress UV sensitivity in recB and recC strains. Also, recombinant plasmids bearing either the sbcB15 or xonA6 allele exhibited a high degree of instability during growth of their bacterial hosts. The results suggest that the sbcB/xonA gene product is a bi- or multifunctional protein that interacts with single-stranded DNA and possibly with other proteins in the suppression of genetic recombination and DNA-repair deficiencies in recB and recC mutants.     

A minor pathway of postreplication repair in Escherichia coli is independent of the recB, recC and recF genes

After ultraviolet (UV) irradiation, an Escherichia coli K12 uvrB5 recB21 recF143 strain (SR1203) was able to perform a limited amount of postreplication repair when incubated in minimal growth medium (MM), but not if incubated in a rich growth medium. Similarly, this strain showed a higher survival after UV irradiation if plated on MM versus rich growth medium (i.e., it showed minimal medium recovery (MMR]. In fact, its survival after UV irradiation on rich growth medium was similar to that of a uvrB5 recA56 strain, which does not show MMR or postreplication repair. The results obtained with a uvrB5 recF332::Tn3 delta recBC strain and a uvrB5 recF332::Tn3 recB21 recC22 strain were similar to those obtained for strain SR1203, suggesting that the recB21 and recF143 alleles are not leaky in strain SR1203. The treatment of UV-irradiated uvrB5 recB21 recF143 and uvrB5 recF332::Tn3 delta recBC cells with rifampicin for 2 h had no effect on survival or the repair of DNA daughter-strand gaps. Therefore, a pathway of postreplication repair has been demonstrated that is constitutive in nature, is inhibited by postirradiation incubation in rich growth medium, and does not require the recB, recC and recF gene products, which control the major pathways of postreplication repair.     

A comparative study of the functions of recBCD-nuclease from Escherichia coli and "recombinase", encoded by plasmid R1drd-19]

The RecBCD nuclease of Escherichia coli and "recombinase" determined by R1drd-19 plasmid (the latter is able to replace at least partially the indicated cellular enzyme) were shown to differ from each other in some essential features. The product encoded by the plasmid as distinct from RecBCD nuclease practically is not sensitive to inhibition by GamS protein of the lambda phage. Earlier, it was found that the presence of R1drd-19 plasmid in the recBC cells restores the level of the total ATP-dependent exonuclease activity because of appearance in such cells of a new exonuclease activity also ATP-dependent. The exonuclease activity determined by R1drd-19 plasmid was found to differ from the corresponding activity of the RecBCD enzyme. The plasmid enzyme was able to prevent reproduction of T4g2- mutant on recBC cells. The ability of the plasmid "recombinase" to some stimulation of intrachromosomal recombination in recA mutant witness to incomplete RecA-dependence of its function. No significant homology was registered between Escherichia coli DNA fragment containing the recB, recC, recD genes and the EcoRI-C-fragment of R1drd-19 carrying the sequences responsible for recombination and repair functions of the plasmid.     

Plasmidic recombination in Escherichia coli K-12: the role of recF gene function

Interplasmidic and intraplasmidic recombination proficiencies were determined in E. coli bacterial strains carrying rec mutations. Our results defined the role of recF gene function, recB, recC, and sbcB gene products (exonuclease V and exonuclease I) in plasmidic recombination in wild-type E. coli cells and in cells in which the recE recombination pathway is activated. RecF gene function is required for interplasmidic recombination regardless of the recB recC genotype. Intraplasmidic recombination is recF dependent in cells having a functional exonuclease V, but not in recB recC mutants. Exonuclease V activity inhibits both interplasmidic and intraplasmidic recombination via the recE pathway.     

Exonucleases I, III, and V are required for stability of ColE1-related plasmids in Escherichia coli.

The stability of two ColE1-related plasmids (pRSF2124 and pMB9) was examined in strains of Escherichia coli multiply deficient in exonucleases I (sbcB), III (xthA), or V (recB recC). Any combination of exonuclease I, III, and V deficiency resulted in dramatically decreased stability of both pRSF2124 and pMB9. Inactivation of the RecF pathway by introducing either recF or recJ mutations to the recB recC subcB background resulted in nearly wild-type levels of stability for both plasmids. In contrast, the introduction of uvrD3 uvr-257, uvrE100, or recL152 into the recB21 recC22 sbcB15 strain did not affect plasmid stability. Furthermore, the amount of plasmid DNA recovered from pRSF2124 or pMB9 transformants of a xthA1 sbcB15 strain was strikingly reduced relative to that of a wild-type control. Taken together, these results suggest that some aspect of DNA repair is required for stable maintenance of ColE1-related plasmids in E. coli.     

Degradation of linear and circular DNA with gaps by the recBC enzyme of Escherichia coli. Effects of gap length and the presence of cell-free extracts

It is shown that circular PM2 DNA with two gaps of 13 nucleotides per molecule is degraded by purified recBC enzyme from Escherichia coli to acid-soluble material at a rate which is less than one tenth of the rate of solubilization of linear duplex DNA. Increasing the gap length in the circular DNA to 40-650 nucleotides does not affect the breakdown of the molecules by the recBC enzyme, nor does it change the proportions of the products formed (acid-soluble material, acid-insoluble fragments and non-degraded molecules). On the other hand, terminal gaps in linear duplex DNA produced by limited digestion with either exonuclease III or lambda exonuclease significantly reduce the rate of the degradation by the recBC enzyme, particularly when the gaps exceed 100 nucleotides. The results suggest that the recBC enzyme does not cleave gaps in circular DNA at random positions, but possibly at the junction between single-stranded and duplex DNA or close to it. The degradation of gapped circular DNA by purified recBC enzyme was used to search for an inhibitor of the recBC enzyme in extracts from ultraviolet-irradiated cells. No such inhibitor has been observed but rather a weak stimulatory factor for the solubilization of gapped circular DNA by the recBC enzyme. Thus, the experimental system appears not to be suited as a test in vitro for an ultraviolet-induced inhibitor of the recBC enzyme which has been postulated to be produced in recA+ lexA+ cells of E. coli after ultraviolet irradiation.     

Genetic Analysis of Mutations Indirectly Suppressing recB and recC Mutations

Mutations in sbcB inactivate exonuclease I and suppress the UV-sensitive, mitomycin-sensitive, recombination-deficient phenotypes associated with recB and recC mutations. Mapping experiments have located sbcB about 0.4 minutes from the his operon at 38.0 on the standard map of E. coli. This places sbcB between supD and his. A four-point cross shows that sbcB lies between P2 attH and his. P2 eduction deleting the his operon beginning with P2 attH also deletes sbcB and produces the expected exonuclease I deficiency and suppression of recB(-). The occurrence of chemical-mutagen-induced and spontaneous mutations indirectly suppressing recB(-) and recC(-) is examined. Three lines of strains produce only sbcA mutations while only sbcB mutations occur in a fourth line. Explanations for this behavior are proposed in light of the ability of the first three lines to express sbcB mutations which they inherit by transduction.     

DNA Structures Generated during Recombination Initiated by Mismatch Repair of Uv-Irradiated Nonreplicating Phage DNA in Escherichia Coli: Requirements for Helicase, Exonucleases, and Recf and Recbcd Functions

During infection of homoimmune Escherichia coli lysogens (``repressed infections'), undamaged non-replicating λ phage DNA circles undergo very little recombination. Prior UV irradiation of phages dramatically elevates recombinant frequencies, even in bacteria deficient in UvrABC-mediated excision repair. We previously reported that 80-90% of this UvrABC-independent recombination required MutHLS function and unmethylated d(GATC) sites, two hallmarks of methyl-directed mismatch repair. We now find that deficiencies in other mismatch-repair activities--UvrD helicase, exonuclease I, exonuclease VII, RecJ exonuclease--drastically reduce recombination. These effects of exonuclease deficiencies on recombination are greater than previously observed effects on mispair-provoked excision in vitro. This suggests that the exonucleases also play other roles in generation and processing of recombinagenic DNA structures. Even though dsDNA breaks are thought to be highly recombinagenic, 60% of intracellular UV-irradiated phage DNA extracted from bacteria in which recombination is low--UvrD(-), ExoI(-), ExoVII(-), or RecJ(-)--displays (near-)blunt-ended dsDNA ends (RecBCD-sensitive when deproteinized). In contrast, only bacteria showing high recombination (Mut(+) UvrD(+) Exo(+)) generate single-stranded regions in nonreplicating UV-irradiated DNA. Both recF and recB recC mutations strikingly reduce recombination (almost as much as a recF recB recC triple mutation), suggesting critical requirements for both RecF and RecBCD activity. The mismatch repair system may thus process UV-irradiated DNA so as to initiate more than one recombination pathway.