Rep Mutants Research Articles

Further characterization of repair of 8-methoxypsoralen crosslinks in UV-excision-defective Escherichia coli

Evidence was previously presented for a new pathway for the repair of 8-methoxypsoralen DNA crosslinks. The pathway, which is independent of the uvrA gene but deficient in rep mutants, has now been further characterized and shown to be more active in minimal than in nutrient growth media and to be inhibited by acriflavine. Although crosslink repair is much reduced in recA bacteria, some still occurs as judged by the effect of acriflavine. By the same criterion, crosslink repair occurs in bacteria with point mutations in the uvrA and uvrB genes, in bacteria with a deletion covering the uvrB gene, and in polA uvrA bacteria. Bacteria with insertions rather than point mutations in the uvrA gene, although showing evidence of repair, demonstrated minimal inhibition with acriflavine suggesting the possibility that the uvrA gene product, even in enzymically inactive, might be able to interact with DNA lesions in the presence of acriflavine and prevent crosslink repair. Crosslink repair in E. coli WP2 uvrA is associated with base-repair substitution mutagenesis and may be characterized as an error-prone process. Crosslink in uvrA bacteria is reduced but not eliminated by a mutation in the umuC gene.

The rep mutation VII. Cloning and analysis of the functional rep gene of Escherichia coli K-12

The rep gene of Escherichia coli was isolated on a 6-kb PvuII fragment of plasmid pLC44-7 DNA from the Clarke-Carbon collection and cloned into pSC101 (to form pHBH8) and pBR322 (to form pHBH3O). The plasmids pHBH8 and pHBH30 were found to complement all rep mutations tested. The functional rep gene and its promoter were mapped to a 3.2-kb XhoI- Ball fragment on the basis of complementation data with deletion and insertion derivatives of the two plasmids; subcloning of various restriction fragments confirmed the assignment. EcoRI, HindIII, and HpaI restriction sites were found to reside within that region of the DNA required for expression of the rep function. A coupled in vitro transcription-translation system was used to show that only those plasmids containing a functional rep gene encoded a protein of about M r 67 000 (the M r of the rep protein). No plasmids were found that complemented only the A or B classes of rep mutants (which differ in their ability to support the growth of P2 and M13 phages). This result suggests that rep-A and rep-B are alleles of the same structural gene.

DNA fusion product of phage P2 with plasmid pBR322: a new phasmid.

The chromosome of the temperate bacteriophage P2 and that of the plasmid pBR322 have been joined in vitro after treatment with restriction endonuclease EcoRI. The fusion product - a phasmid - can behave as a plasmid, as a phage and as a prophage. It can replicate its DNA under the control of either the specific replication mechanism of the parent phage in a polA mutant or that of the parent plasmid in a rep mutant. Several interesting interactions between the two replication modes are indicated. In particular, phage particles may be produced even when the phage mode of DNA replication is blocked, and this throws new light on the involvement of the early gene A in the regulation of late gene expression in phage P2.

A new pathway for repair of cross-linkable 8-methoxypsoralen mono-adducts in Uvr − strains of Escherichia coli

UvrA-deficient Echerichia coli bacteria possess a DNA repair pathway for certain photoinduced mono-adducts of 8-methoxypsoralen. Such adducts can be converted to highly lethal cross-links by a second exposure to near-UV light in the absence of 8-methoxypsoralen. If a period of incubation is allowed to intervene between the primary and cross-linking exposure, the lethal effect is considerably diminished, indicating that some of the mono-adducts become unavailable for cross-linking. A repair pathway is postulated which is pre-replicative, unaffected by chloramphenicol, acriflavine or caffeine, and inhibited at 0°C. It is unaffected by a rep mutation but is blocked by a polA mutation, suggesting that it involves DNA strand breakage and at least some exonuclease action.

Replication of M13 oriC bacteriophages in Escherichia coli rep mutant is dependent on the cloned Escherichia coli replication origin.

The involvement of the Escherichia coli rep protein in the replication of M13 chimeric deoxyribonucleic acids (DNAs) carrying the E. coli chromosomal DNA replication origin (oriC) has been examined. Previous studies indicate that the cloning of a 3,550-base-pair sequence of chromosomal DNA containing oriC into an M13 vector allows extensive replication of the M13 oriC chimeric DNA in an E. coli rep-3 mutant. We have extended these studies by preparing a 330-base-pair deletion that specifically deletes the oriC sequence in the M13 oriC DNAs, to demonstrate that the replication observed in the rep-3 host is dependent on the cloned origin. Thus, a DNA-unwinding enzyme other than the rep protein may be involved in the strand separation process accompanying replication which initiates at oriC in the M13 oriC chimeric DNAs and in the E. coli chromosome. The rep assay used for assessing the functionality of the cloned oriC is useful for analysis of any rep-independent origin of replication functional in E. coli. A direct selection for a cloned origin of replication is possible in the rep-3 recA56 host. Since the cloned origin is nonessential for propagation of the M13 chimeric phage in a rep+ host, mutations in the cloned origin may be constructed, and the mutant phage may be examined by a simple transductional analysis of the rep-3 recA56 mutant strain.

Identification of a ØX174 coded protein involved in the inhibit on of β-galactosidase synthesis in [formula omitted

The synthesis of β-galactosidase (EC 3.2.1.23: β-D-galactoside galactohydrolase) in Escherichia coli is repressed as a result of infection with single-stranded DNA phage ØX174. An amber mutant in ØX174 cistron A, which codes for two proteins, does not inhibit the enzyme synthesis while amber mutants in all other genes do cause repression. A mutant near the amino-terminal end of cistron A, which produces the small 35,000 molecular weight cistron A polypeptide, also inhibits the synthesis of β-galactosidase. Inhibition is also observed in an Escherichia coli rep mutant which does not support the replication of replicative-form DNA. Exogenous nucleotide bases and cyclic 3′,5′-adenosine monophosphate (cyclic AMP) do not have any effect on the degree of repression.

Replication mutants of the F-plasmid of Escherichia coli K-12 : I. Isolation and characterization of temperature-sensitive replication mutants of F′- gal+

Eighteen mutants that are temperature-sensitive for vegetative replication ( rep) were isolated from two F′- gal + plasmids (F8 and F8-4) after N-methyl- N′-nitro- N-nitrosoguanidine mutagenesis. Some of the mutants also have reduced transfer ability at both permissive and nonpermissive temperatures. Plasmid-plasmid P1 transduction has revealed that in some instances, the altered transfer ability is located in the transfer operon and is distinct from the rep mutation. However, in other cases, the replication and transfer defects have not been separated by P1 transduction. The implications of these results for the relationship between vegetative DNA replication and DNA replication during conjugation are discussed. In vivo recombinational results suggested that the temperature-sensitive mutations were not located in the same regions of the two F′-plasmids. We confirmed that no inversion, secondary deletion, or translocation of DNA had occurred in either F8 or F8-4, and suggest that the apparent difference is due to a recombination anomaly.

Control of plasmid R6K copy numbers in isogenic rep+ and rep Escherichia coli strains.

We have analysed as a function of cell doubling times the control of R6K plasmid replication in rep+ and rep strains of Escherichia coli. The rep mutation results in an alteration or loss of an enzyme that unwinds helical DNA. We found in rep+ bacteria that R6K relative dosage (plasmids per genome equivalent) remained nearly constant as growth rates increased. From this we concluded that the average plasmid concentration (plasmids per unit cell mass or volume) fell relative to the average concentration of chromosome origins when growth rates increased. In this context, the control of R6K replication is similar to that of other plasmids as seen by different workers. We also found that the relative dosage of R6K in rep mutants is greater than in rep+ bacteria when both strains were grown at fast growth rates. This finding was expected since at fast growth rates the number of genome equivalents per unit mass is expected to be lower in rep mutants. Unexpectedly, however, we found the effect of the rep mutation on R6K relative dosage had occurred in a step-like manner at a slow growth rate of about 120 min per generation. This implies that both the relative dosage and concentration of R6K had increased in a step-like manner. We also found that the effect of the rep mutation on R6K concentration was lost at fast growth rates while the effect of the mutation on R6K relative dosage was not lost.

The rep mutation. VI. Purification and properties of the Escherichia coli rep protein, DNA helicase III

The protein product of the rep gene of Escherichia coli is required for the replication of certain bacteriophage genomes (phi X174, fd, P2) and for the normal replication of E. coli DNA. We have used a specialized transducing phage, lambda p rep+, which complements the defect of rep mutants, to identify the rep protein. The rep protein has been purified from cells infected with lambda p rep+ phage; it has a molecular weight of about 70 000 and appears similar to the protein found in normal cells. Stimulation of phi X174 replicative form DNA synthesis in vitro was observed when highly purified rep protein was supplied to a cell extract derived from phi X-infected E. coli rep cells and supplemented with replicative form DNA. The purified protein has a single-stranded DNA-dependent ATPase activity and is capable of sensitizing duplex DNA to nucleases specific for single-stranded DNA. For this reason we propose the enzyme be called DNA helicase III. We infer that the rep protein uses the energy of hydrolysis of ATP to separate the strands of duplex DNA; the E. coli DNA binding protein need not be present. The rep3 mutant appeared to make a limited amount of active rep protein.

“Nick translation” in Escherichia coli rep strains deficient in DNA polymerase I activities

Using phiX1974 replicative form (RF) DNA as an in vivo probe, we have investigated the coordinated action of the 5' leads to 3' exonuclease and polymerase activities of DNA polymerase I in order to understand better its physiological role. We constructed double mutants containing the rep mutation (the replication of phiX174 RF does not occur in rep mutants) together with a mutation affecting DNA polymerase I, either polA12 or polA546ex. Using these mutants, which are believed to be thermosensitive in the polymerase function or the 5' leads to 3' exonuclease function respectively, we studied the kinetics of nick translation at the permissive and non-permissive temperatures in vivo. The substrate was the phiX174 replicative form DNA nicked by the phiX174 gene A protein. E. coli rep polA546ex gave the lowest rate of nick translation, although the ability to perform nick translation, at least as measured by our assay, was still present. E. coli rep polA12 showed a similar low rate at the non-permissive temperature but a rate close to the wild-type level at the permissive temperature. Formation of the parental replicative form molecule in either strain was affected little, even at the restrictive temperature. Our results suggest that DNA polymerase I may not play a major role in ongoing DNA replication.

Recombinant levels of Escherichia coli K-12 mutants deficient in various replication, recombination, or repair genes

Escherichia coli strains containing mutations in lexA, rep, uvrA, uvrD, uvrE, lig, polA, dam, or xthA were constructed and tested for conjugation and transduction proficiencies and ability to form Lac+ recombinants in an assay system utilizing a nontandem duplication of two partially deleted lactose operons (lacMS286phi80dIIlacBK1). lexA and rep mutants were as deficient (20% of wild type) as recB and recC strains in their ability to produce Lac+ progeny. All the other strains exhibited increased frequencies of Lac+ recombinant formation, compared with wild type, ranging from 2- to 13-fold. Some strains showed markedly increased conjugation proficiency (dam uvrD) compared to wild type, while others appeared deficient (polA107). Some differences in transduction proficiency were also observed. Analysis of the Lac+ recombinants formed by the various mutants indicated that they were identical to the recombinants formed by a wild-type strain. The results indicate that genetic recombination in E. coli is a highly regulated process involving multiple gene products.

Synthesis of α3 phage DNA in replication mutants of Escherichia coli

Host functions for DNA replication of bacteriophage α3, a representative of group A microvirid phages, were studied using dna and rep mutants of Escherichia coli. In dna + cells, conversion of phage α3 single-stranded DNA (SS) into the double-stranded replicative form (RF) was insensitive to 30–150 μg/ml of chloramphenicol, 200 μg/ml of rifampicin, 50 μg/ml of nalidixic acid, or 200 μg/ml of novobiocin. At 43°C, synthesis of the parental RF was inhibited in dnaG and dnaZ mutants, but not in dnaE and rep strains. Replication of phage α3 progeny RF was prevented by 50 μg/ml of mitomycin C (in hcr + bacteria), 50 μg/ml of nalidixic acid or 200 μg/ml of novoviocin, but neither by 30 μg/ml of chloramphenicol nor by 200 μg/ml of rifampicin. Besides dnaG and dnaZ gene products, dnaE and rep functions were essential for the progeny RF synthesis. Host factor dependence of α3 was relatively simple and, in contrast with phages øX174 and G4, α3 did not require dnaB and dnaC(D) activities.

The rep mutation : IV. Slower movement of replication forks in Escherichia coli rep strains

The rep gene function of Escherichia coli is essential for the replication of bacteriophage P2 and X174 double-stranded DNA. In addition, the replicating bacterial chromosome in E. coli rep strains differs from that of wild-type strains in that it contains more replication forks. We have done various experiments to analyze the effect of the rep function on replication fork movement. (1) When cells grown in heavy-density (2H, 15N) medium are shifted to light (1H, 14N) medium and their DNA analyzed by CsCl density-gradient centrifugation, the rate at which heavy DNA is transferred to the hybrid DNA position is found to be slower in the rep mutant than in the wild type. (2) Grain tracks produced by autoradiography of pulse-labeled DNA from a rep strain are shorter than those from wild-type cells. (3) Replicating chromosomes in the rep mutant show a steeper gradient of marker frequencies than those in the rep+ strain. We conclude from the results that in rep mutants the speed of replication fork movement is only 50 to 60% of that of wild-type cells.

The rep mutation. III. Altered structure of the replicating Escherichia coli chromosome.

The rep gene function of Escherichia coli is essential for the replication of P2 and phiX174 double-stranded deoxyribonucleic acid (DNA). Compared with isogenic rep(+) strains, rep mutants show the following characteristics: larger cell size, more DNA per cell, and a slightly lower DNA/mass ratio. The replicating rep chromosomes show a steeper gradient of marker frequencies and contain more replicating forks per chromosome. The nucleoid body of rep mutants sediments faster and contains more DNA. We deduce that the rep function is required for the "normal" replication of the E. coli chromosome and that in its absence the E. coli chromosome replicates in an altered manner, perhaps involving slower-moving replicating forks.

Replicator mutants of bacteriophage λ: Characterization of two subclasses

Further studies on one class of mutations of bacteriophage λ ( Rambach and Brachet, 1971) are reported in this paper. A prophage carrying such a mutation does not replicate, and the effect is cis. The mutations, called rep, are located between the CII and O genes. Among nine rep mutations, two subclasses can be distinguished on the basis of genetic results.

The rep mutation : II. Its effect on Escherichia coli and on the replication of bacteriophage ∅X174

Escherichia coli REP − mutants infected with bacteriophage ∅X174 are able to incorporate the single-stranded viral DNA into the ∅X replicative form (RF); both RF I (superhelix) and RF II are formed but there is relatively more RF II formed in the mutant than in REP + cells. The RF does not replicate, although it is able to associate with a presumed membrane site at which DNA replication is thought to occur. Transcription of the viral genome takes place, albeit in reduced amounts, and all phage proteins are made. The mutant has an increased sensitivity to X-rays and a somewhat reduced capacity to support bacterial recombination; contrary to earlier claims this deficiency is slight and may be the result of a generalized impairment of the cellular metabolism. The existence of rep mutations suppressible by amber suppressors suggests that the functional product of the rep + gene is a protein.

Characterization of REP − mutants and their interaction with P2 phage

Three rep 5 5 Abbreviations: rep, a mutation which affects DNA replication of ∅X174 (Denhardt et al., 1967). The nomenclature of Denhardt et al. has been modified to conform to the recommendations of Demerec et al. (1966). In Denhardt's nomenclature REP − n described a mutation and also served as the name of the strain carrying that mutation. In this paper REP + and REP − are used to designate phenotypes, rep + and rep are used for genotypes, and rep-n is used to designate rep mutation number. NG, N-methyl- N-nitrosoguanidine. PFU, plaque-forming unit(s). UV, ultraviolet light. Other abbreviations are explained in footnote a to Table 1. mutations in Escherichia coli have been mapped between metE and ilv. rep is recessive. Using REP − strains derived by transduction, we have obtained no evidence that three rep mutations confer recombination deficiency, although they do do appear to confer a slight increase in UV sensitivity. A number of temperate coliphages cannot use REP − mutants as a host in lytic multiplication. The interaction of REP − mutants and the non-inducible temperate phage P2 is examined in detail, with the following findings: (1) P2 can inject its DNA into REP − hosts, and the phage DNA can circularize, but replication of the DNA is blocked. (2) P2 can lysogenize REP − mutants at approximately normal frequency. (3) REP − mutants are resistant to killing by P2.

Bacterial control of λ replication: I. Initial characterization of a rep mutation of Escherichia coli K12

There is evidence to suggest that host cells catalyze several of the steps in the early stages of infection of coliphage λ. From C600, a λ-sensitive strain of E. coli K12, a class of bacterial mutants has been isolated which fail to replicate the λ genome; these mutants have been tentatively named repI. One representative mutant ( repI21) has been studied further. The physiology and mating efficiency of the mutant strain appeared to be relatively unaffected by the mutation. The principal characteristic of the bacterial mutant, as compared to C600, was the inability of λ to undergo the normal cycle of lytic development in this mutant strain, though evidence in support of λ integration within the host chromosome had been provided by an increased frequency of λ transduction of the galactose (gal) gene of E. coli. The repI21 mutation inhibited the growth of wild-type λ ( λ +), λC I, λb2 and all hybrids of λimm 434, λimm 21, λimm ∅80. Although the mechanism of control of λ replication in strain repI21 is not clearly understood, a genetic control due to some structural defect in the region of λ integration or to an enzymatic block involving an early protein necessary for λ replication is inferred.