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.     

Genetic and Physical Analysis of Plasmid Recombination in Recb Recc Sbcb and Recb Recc Sbca Escherichia Coli K-12 Mutants

The effect of mutations in known recombination genes (recA, recB, recC, recE, recF, recJ, recN, recO, recQ and ruv) on intramolecular recombination of plasmids was studied in recB recC sbcB and recB recC sbcA Escherichia coli mutants. The rate of recombination of circular dimer plasmids was at least 1000-fold higher in recB recC sbcB or recB recC sbcA mutants as compared to wild-type cells. The rate was decreased by mutations in recA, recF, recJ, recO, ruv or mutS in recB recC sbcB mutants, and by mutations in recE, recN, recO, recQ, ruv or mutS in recB recC sbcA mutants. In addition to measuring the recombination rate of circular dimer plasmids, the recombination-mediated transformation of linear dimer plasmids was also studied. Linear dimer plasmids transformed recB recC sbcB and recB recC sbcA mutants 20- to 40-fold more efficiently than wild-type cells. The transformation efficiency of linear dimer plasmids in recB recC sbcB mutants was decreased by mutations in recA, recF, recJ, recO, recQ or lexA (lexA3). In recB recC sbcA mutants the transformation efficiency of linear dimers was decreased only by a recE mutation. Physical analysis of linear dimer- or circular dimer-transformed recB recC sbcB mutants revealed that all transformants contained recombinant monomer genotypes. This suggests that recombination in recB recC sbcB cells is very efficient.     

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.     

Gene conversion in Escherichia coli: the recF pathway for resolution of heteroduplex DNA.

The independent repair of mismatched nucleotides present in heteroduplex DNA has been used to explain gene conversion and map expansion after general genetic recombination. We have constructed and purified heteroduplex plasmid DNAs that contain heteroallelic 10-base-pair insertion-deletion mismatches. These DNA substrates are similar in structure to the heteroduplex DNA intermediates that have been proposed to be produced during the genetic recombination of plasmids. These DNA substrates were transformed into wild-type and mutant Escherichia coli strains, and the fate of the heteroduplex DNA was determined by both restriction mapping and genetic tests. Independent repair events that yielded a wild-type Tetr gene were observed at a frequency of approximately 1% in both wild-type and recB recC sbcB mutant E. coli strains. The independent repair of small insertion-deletion-type mismatches separated by 1,243 base pairs was found to be reduced by recF, recJ, and ssb single mutations in an otherwise wild-type genetic background and reduced by recF, recJ, and recO mutations in a recB recC sbcB genetic background (the ssb mutation was not tested in the latter background). Independent repair of small insertion-deletion-type mismatched nucleotides that were as close as 312 nucleotides apart was observed. There was no apparent bias in favor of the insertion or deletion of mutant sequences.     

Effect of transient lambda prophage induction on ultraviolet light resistance and recombination in Escherichia coli.

Transient induction of lambda prophage increases the ultraviolet light resistance of most exponentially growing Escherichia coli lysogens. Resistance is increased in wild-type, recB, recB recC, recB recC recF, and recB recC recL hosts. No enhancement in recA lysogens was found, nor was there enhancement in stationary cultures. Enhancement was dependent upon the lambdared recombination system. Transient induction also increases the genetic recombination rate in recB lysogens as measured in Hfr X F- matings.     

recA (Srf) suppression of recF deficiency in the postreplication repair of UV-irradiated Escherichia coli K-12.

The mechanism by which recA (Srf) mutations (recA2020 and recA801) suppress the deficiency in postreplication repair shown by recF mutants of Escherichia coli was studied in UV-irradiated uvrB and uvrA recB recC sbcB cells. The recA (Srf) mutations partially suppressed the UV radiation sensitivity of uvrB recF, uvrB recF recB, and uvrA recB recC sbcB recF cells, and they partially restored the ability of uvrB recF and uvrA recB recC sbcB recF cells to repair DNA daughter-strand gaps. In addition, the recA (Srf) mutations suppressed the recF deficiency in the repair of DNA double-strand breaks in UV-irradiated uvrA recB recC sbcB recF cells. The recA2020 and recA801 mutations do not appear to affect the synthesis of UV radiation-induced proteins, nor do they appear to produce an altered RecA protein, as detected by two-dimensional gel electrophoresis. These results are consistent with the suggestion (M. R. Volkert and M. A. Hartke, J. Bacteriol. 157:498-506, 1984) that the recA (Srf) mutations do not act by affecting the induction of SOS responses; rather, they allow the RecA protein to participate in the recF-dependent postreplication repair processes without the need of the RecF protein.     

Suppression of Escherichia coli recF mutations by recA-linked srfA mutations.

Suppressors of recF (srfA) were found by selection for resistance to mitomycin C and UV irradiation in a recB21 recC22 sbcB15 recF143 strain. srfA mutations map in recA and are dominant to srfA+. They suppress both the DNA repair and the recombination deficiencies due to recF mutations. Therefore, RecA protein which is altered by the srfA mutation can allow genetic recombination to proceed in the absence of recB, recC, and recF functions. recF is also required for induction of the SOS response after UV damage. We propose that recF+ normally functions to allow the expression of two recA activities, one that is required for the RecF pathway of recombination and another that is required for SOS induction. The two RecA activities are different and are separable by mutation since srfA mutations permit recombination to proceed but have not caused a dramatic increase in SOS induction in recF mutants. According to this hypothesis, one role for recF in DNA repair and recombination is to modulate RecA activities to allow RecA to participate in these recF-dependent processes.     

Reactivation of ultraviolet-irradiated phage lambda and recombination in Escherichia coli K-12 cells containing plasmid ColIb-P9

It was shown that the presence of colicinogenis plasmid ColIb-P9 increased the survival of UV-irradiated bacteriophage lambda cI857 in non-irradiated cells of Escherichia coli K-12. The effect of this plasmid was retained in the polA and recB mutants, being sharply reduced in the uvrA and recB recC sbcB recF mutants. This effect strongly depended on recA+ and lexA+ genotype. The W-reactivation efficiency was slightly higher in the cells containing ColIb-P9 than in those lacking the plasmid. No significant effect of the plasmid on recombination during transduction, after conjugation under usual conditions and in the case when a conjugation mixture or recipient cells were irradiated, was observed. The data demonstrate that the effect of ColIb-P9 plasmid on DNA repair is not mediated by its influence on recombination.     

Different effects of recJ and recN mutations on the postreplication repair of UV-damaged DNA in Escherichia coli K-12.

Two mutations known to affect recombination in a recB recC sbsBC strain, recJ284::Tn10 and recN262, were examined for their effects on the postreplication repair of UV-damaged DNA. The recJ mutation did not affect the UV radiation sensitivity of uvrB and uvrB recF cells, but it increased the sensitivity of uvrB recN (approximately 3-fold) and uvrB recB (approximately 8-fold) cells. On the other hand, the recN mutation did not affect the UV sensitivity of uvrB recB cells, but it increased the sensitivity of uvrB (approximately 1.5-fold) and uvrB recF (approximately 4-fold) cells. DNA repair studies indicated that the recN mutation produced a partial deficiency in the postreplication repair of DNA double-strand breaks that arise from unrepaired daughter strand gaps, while the recJ mutation produced a deficiency in the repair of daughter strand gaps in uvrB recB cells (but not in uvrB cells) and a deficiency in the repair of both daughter strand gaps and double-strand breaks in uvrA recB recC shcBC cells. Together, these results indicate that the recJ and recN genes are involved in different aspects of postreplication repair.     

Analysis of genetic recombination between two partially deleted lactose operons of Escherichia coli K-12.

Genetic recombination between a nontandem duplication of two partially deleted lactose operons (lacMS286phi80dIIlacBK1) in Escherichia coli K-12 has been examined. Since the deletions were nonoverlapping, rare lactose-fermenting (Lac+) recombinants occurred and were detected qualitatively on lactose tetrazolium agar indicator plates as white papillae growing on the surface of red colonies or quantitively on lactose minimal agar plates. Formation of Lac+ recombinants required the recA, recB, and recC gene products. Indirect suppression of recB21 by sbcB15 led to an increase in the frequency of Lac+ recombinants over wild-type levels. recF143 did not appreciably alter the number of Lac+ progeny, whereas recL152 and sbcB15 strains yielded increased numbers of Lac+ recombinants. The nature and formation of Lac+ recombinants was also examined. Respreading analysis indicated that formation of recombinants occurred primarily as the cells entered early stationary phase on the surface of the minimal agar plates and that over 90% of the recombinants contained a phi80dIIlac+ prophage. Time-of-entry experiments suggested that the region of deoxyribonucleic acid between the two operons was not inverted as a result of the recombinational event.     

Binary mixtures containing isomers of nitrobenzo[a]pyrene induce greater-than-additive mutational responses in Salmonella typhimurium. I. Analysis by the total concentration-proportional mixture model

The inhibition of cell division induced by bleomycin (BM) and UV irradiation in the set of rec mutants of E. coli K12 was studied. Data presented in this work indicate that BM treatment requires mainly the RecBC pathway for the induction of cell filamentation. In the recB21 mutant cell filamentation is delayed and reduced compared to the wild type. Cell filamentation is BM-induced with similar kinetics in strains with a proficient RecBC recombination pathway (rec+, recF143 and recN262), as well as in the strain with a fully expressed RecF pathway (recB21recC22sbcB15). Induction is completely abolished in the recB21recF143 double mutant. On the other hand cell filamentation was induced similarly by UV irradiation in all strains with a functional recF gene and in the strain with a fully operative RecF pathway, but it was delayed in the recF143 and recB21recF143 mutants.     

Effects of the Escherichia coli recF suppressor mutation, recA801, on recF-dependent DNA-repair associated phenomena

In recb recC sbcB mutants genetic recombination is dependent upon the recF gene. recA801, recA802 and recA803 (formerly called srfA mutations) were originally isolated as mutations that suppress recombination deficiency caused by a recF mutation in a recB recC sbcB genetic background. Since the recA801 mutation also suppressed some of the UV sensitivity due to recF143, we sought to determine what DNA-repair pathways were actually being restored by the recA801 mutation in this genetic background. In this paper we show that the suppression of recF143 by recA801 does not extend to the recF143-mediated defects in induced repair of UV-damaged phages. In addition, we show that recA801 suppresses only slightly the recF143-associated defect in induced expression of the SOS-regulated muc genes of pKM101. These results suggest that recA801 suppresses primarily the RecF pathway of recombinational repair.     

sbcB sbcC null mutations allow RecF-mediated repair of arrested replication forks in rep recBC mutants

We have proposed previously that, in Escherichia coli, blockage of replication forks can lead to the reversal of the fork. Annealing of the newly synthesized strands creates a double-stranded end adjacent to a Holliday junction. The junction is migrated away from the DNA end by RuvAB and can be cleaved by RuvC, while RecBCD is required for the repair of the double-stranded tail. Consequently, the rep mutant, in which replication arrests are frequent and fork reversal occurs, requires RecBCD for growth. We show here that the combination of sbcB sbcCD null mutations restores the viability to rep recBC mutants by activation of the RecF pathway of recombination. This shows that the proteins belonging to the RecF pathway are able to process the DNA ends made by the replication fork reversal into a structure that allows recombination-dependent replication restart. However, we confirm that, unlike sbcB null mutations, sbcB15, which suppresses all other recBC mutant defects, does not restore the viability of rep recBC sbcCD strains. We also show that ruvAB inactivation suppresses the lethality and the formation of double-stranded breaks (DSBs) in a rep recBC recF strain, totally deficient for homologous recombination, as well as in rep recBC mutants. This confirms that RuvAB processing of arrested replication forks is independent of the presence of recombination intermediates.     

Survival of recombination-deficient mutants of Escherichia coli during incubation with nalidixic acid.

The ability of several Escherichia coli strains deficient in recombination (rec) to survive in the presence of nalidixic acid was determined. Genetic blocks of the RecBC or the RecF pathways resulted in increased sensitivity to nalidixic acid when compared with the wild-type strain. Mutants lacking functional recA, recL, or recB recC recF genes showed the most rapid decrease in colony-forming ability when incubated with nalidixic acid. However, the uvrB gene also plays a role in maintaining cell viability.     

Synthesis of linear plasmid multimers in Escherichia coli K-12.

Linear plasmid multimers were identified in extracts of recB21 recC22 strains containing derivatives of the ColE1-type plasmids pACYC184 and pBR322. A mutation in sbcB increases the proportion of plasmid DNA as linear multimers. A model to explain this is based on proposed roles of RecBC enzyme and SbcB enzyme (DNA exonuclease I) in preventing two types of rolling-circle DNA synthesis. Support for this hypothesis was obtained by derepressing synthesis of an inhibitor of RecBC enzyme and observing a difference in control of linear multimer synthesis and monomer circle replication. Reinitiation of rolling-circle DNA synthesis was proposed to occur by recA+-dependent and recA+-independent recombination events involving linear multimers. The presence of linear plasmid multimers in recB and recC mutants sheds new light on plasmid recombination frequencies in various mutant strains.     

Genetic analysis of the recG locus of Escherichia coli K-12 and of its role in recombination and DNA repair.

We describe a transposon insertion that reduces the efficiency of homologous recombination and DNA repair in Escherichia coli. The insertion, rec-258, was located between pyrE and dgo at min 82.1 on the current linkage map. On the basis of linkage to pyrE and complementation studies with the cloned rec+ gene, rec-258 was identified as an allele of the recG locus first reported by Storm et al. (P. K. Storm, W. P. M. Hoekstra, P. G. De Haan, and C. Verhoef, Mutat. Res. 13:9-17, 1971). The recG258 mutation confers sensitivity to mitomycin C and UV light and a 3- to 10-fold deficiency in conjugational recombination in wild-type, recB recC sbcA, and recB recC sbcB sbcC genetic backgrounds. It does not appear to affect plasmid recombination in the wild-type. A recG258 single mutant is also sensitive to ionizing radiation. The SOS response is induced normally, although the basal level of expression is elevated two- to threefold. Further genetic studies revealed that recB recG and recG recJ double mutants are much more sensitive to UV light than the respective single mutants in each case. However, no synergistic interactions were discovered between recG258 and mutations in recF, recN, or recQ. It is concluded that recG does not fall within any of the accepted groups of genes that affect recombination and DNA repair.     

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.     

Effect of mutations in recB, recC, sbcB and recF genes controlling the homologous recombination in Escherichia coli cells on transposition of Tn1

The mutations in the genes controlling the homologous DNA recombination in Escherichia coli cells effect the efficiency of Tn1 transposition. Mutations in recB and recC genes decrease 50-fold the frequencies of Tn1 transposition. Introduction of an additional mutation in sbcB gene increase transposition frequency for three orders as compared with the one registered in wild type cells. Inactivation of sbcB gene in the wild type cells does not affect transposition significantly. Mutation in recF gene results in the great decrease of transposition when it is introduced into multiple recBC sbcB mutant, but not into the wild type bacteria. The possibility of two pathways for Tn1 transposition existing in Escherichia coli cells is discussed, as well as possibility of existence of similar stages in transposition and recombination controlled by the same genes.     

Plasmid control of recombination in E. coli K 12

Summary The recombination proficiency of three recipient strains of Escherichia coli K 12 carrying different plasmids was investigated by conjugal mating with Hfr Cavalli. Some plasmids (e.g. R1drd 19, R6K) caused a marked reduction in the yield of recombinants formed in crosses with Hfr but did not reduce the ability of host strains to accept plasmid F104. The effect of plasmids on recombination was host-dependent. In Hfr crosses with AB1157 (R1-19) used as a recipient the linkage between selected and unselected proximal markers of the donor was sharply decreased. Plasmid R1-19 also decreased the yield of recombinants formed by recF, recL, and recB recC sbcA mutants, showed no effect on the recombination proficiency of recB recC sbcB mutant, and increased the recombination proficiency of recB, recB recC sbcB recF, and recB recC sbcB recL mutants. An ATP-dependent exonuclease activity was found in all tested recB recC mutants carrying plasmid R1-19, while this plasmid did not affect the activity of exonuclease I in strain AB1157 and its rec ^– derivatives. The same plasmid was also found to protect different rec ^– derivatives of the strain AB1157 against the lethal action of UV light. We suppose that a new ATP-dependent exonuclease determined by R1-19 plays a role in both repair and recombination of the host through the substitution of or competition with the exoV coded for by the genes recB and recC.     

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.