Replication of plasmid R6K in DNA polymerase deficient mutants of Escherichia coli.

The plasmid R6K has been introduced into a number of Escherichia coli polymerase deficient (pol) mutants. In polCts mutants transferred to the nonpermissive temperature to inactivate polymerase III, R6K replicates but the replication products have a density in dye-CsCl gradients intermediate between supercoiled and linear forms. This aberrant replication requires normal cellular levels of polymerase I since it does not occur in polA polCts mutants. Normal R6K replication and maintenance occur in a polA polB polC+ host, however, we cannot tell from our experiments wheather polymerase I or III replicates R6K in polA+ polC+ host. Polymerase II, the polB gene product, has no detectable role in R6K replication.     

HU-1 mutants of Escherichia coli deficient in DNA binding

We constructed four mutants of the Escherichia coli hupB gene, encoding HU-1 protein, by synthetic oligodeoxyribonucleotide-directed, site-specific mutagenesis on M13mp18 vectors. The HupBR45 protein contained alterations of Arg58----Gly and Arg61----Gly, and the HupBF3, HupBK2 and HupBA1 proteins contained Phe47----Thr, Lys37----Gln and Ala30----Asp alterations, respectively. HupBF3 and HupBR45 were unable to maintain normal cell growth in a hupA-hupB-himA triple mutant at 42 degrees C, mini-F or RSF1010 proliferation, or Mu phage development in a hupA-hupB double mutant, whereas HupBA1 and HupBK2 supported these cellular activities. DNA-affinity column chromatography showed that the HupBF3 and HupBR45 had reduced affinities to DNA. These observations indicate that two highly conserved Arg residues in the arm structure of the C-terminal half of the HU-1 molecule and a Phe residue in the short beta-sheet connecting the two halves of the molecule are important for the DNA-binding ability and biological functions of this protein.     

Prophage induction in Escherichia coli K12 cells deficient in DNA polymerase I.

Summary The induction of prophage by ultraviolet light has been measured inE. coli K12 lysogenic cells deficient in DNA polymerase I. The efficiency of the induction process was greater inpolA1 polC(dnaE) double mutants incubated at the temperature that blocks DNA replication than inpolA ⁺ polC single mutants. Similarly, thepolA1 mutation sensitizedtif-promoted lysogenic induction in apolA1 tif strain at 42°. In strains bearing thepolA12 mutation, which growth normally at 30°, induction of the prophage occured after the shift to 42°. It is concluded that dissapearance of the DNA polymerase I activity leads to changes in DNA replication that are able, per se, to trigger the prophage induction process.     

Ribosomal abnormality in recA mutants of Escherichia coli.

Summary The tif-1 mutation has been shown to affect protein synthesis in vitro by increasing translational ambiguity (Ephrati-Elizur, Luther-Davies and Hayes, 1976). It is demonstrated here that some recA mutations confer similar abnormality. By comparing suitable combinations of ribosomes and soluble proteins from recA ⁺ and recA cells the defect is shown to be associated with ribosomes. The recA mutation, which suppresses most phenotypic characteristics of the tif-1 mutation (Castellazzi, George and Buttin, 1972(b)) does not suppress the ribosomal abnormality. Sience the closely linked tif-1 and recA mutations lead to the expression of a common property they may be in the same gene.     

Bacteriophage T4 mutants deficient in alteration and modification of the Escherichia coli RNA polymerase

An extensive screening of coliphage T4 mutants has revealed two distinct classes defective, respectively, in the two sequential phage-induced phosphorylations of the host RNA polymerase, alteration and modification. The existence of these mutants proves that T4-specified functions are involved in both processes. The viabilities of these mutants demonstrate that neither alteration nor modification is essential for growth in Escherichia coli B/r. Physiological studies after infection of E. coli B/r have failed to reveal any abnormalities of phage deficient in alteration or modification. Both mutants normally inhibit host protein and stable RNA synthesis and normally express all classes of T4 genes. Thus, these specific phage-induced structural changes in the host RNA polymerase are not fundamental to the control of gene expression during T4 development. Alteration and modification may be required for growth in some strains of E. coli and hence be selectively advantageous because they extend the normal host range of the phage.Alteration appears to be catalyzed by a T4 function injected with the DNA. A polypeptide of molecular weight 61,000, which is probably cleaved during morphogenesis from a precursor of molecular weight 79,000, is missing in phage particles of alteration-deficient strains and may be the phage activity so injected. The T4 gene involved in alteration is named alt.Modification is controlled by a T4-replicative gene that has been mapped into a region of about 500 base-pairs between genes 39 and 56. These mapping data show that the defect in α modification defines a new T4 gene, named mod.     

Conditional impairment of cell division and altered lethality in hipA mutants of Escherichia coli K-12.

Mutations in hipA, a gene of Escherichia coli K-12, greatly reduce the lethality of selective inhibition of peptidoglycan synthesis. These mutations have also been found to reduce the lethality that accompanies either selective inhibition of DNA synthesis or heat shock of strains defective in htpR. In addition, the mutant alleles of hipA are responsible for a reversible cold-sensitive block in cell division and synthesis of macromolecules, particularly peptidoglycan. Recombination between the chromosome of hipA mutants and plasmids containing noncomplementing fragments of hipA+ revealed that the mutations responsible for both cold sensitivity and reduced lethality were probably identical and, in any case, lay within the first 360 base pairs of the coding region of hipA, probably within the first 50 base pairs. We suggest that the pleiotropic effects of mutations in hipA reflect the involvement of this gene in cell division.     

Influence of growth phase on UV induced lethality and DNA breakdown in a Kornberg polymerase deficient resA strain of Escherichia coli

Summary A stationary phase culture of the Kornberg polymerase deficient resA strain R15 was more sensitive to UV irradiation than a growing culture. The dose reduction factor was 2–2.5. UV induced DNA breakdown was also greater in a stationary culture than in a growing culture. The relationship between lethality and DNA breakdown was not quantitative. On the basis of these results we suggest that in this DNA polymerase deficient mutant the repolymerisation step of excision repair may take place at or after DNA replication. Survival and DNA breakdown after UV would then depend in a complex way on the resultant of the rates of excision, nuclease activity and DNA replication.     

"Nick translation" in Escherichia coli rep strains deficient in DNA polymerase I activities.

Summary Using X174 replicative form (RF) DNA as an in vivo probe, we have investigated the coordinated action of the 53 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 X174 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 53 exonuclease function respectively, we studied the kinetics of nick translation at the permissive and non-permissive temperatures in vivo. The substrate was the X174 replicative form DNA nicked by the X174 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 wildtype 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.     

DNA polymerase I and the bypassing of RecA dependence of constitutive stable DNA replication in Escherichia coli rnhA mutants.

In Escherichia coli rnhA mutants, several normally repressed origins (oriK sites) of DNA replication are activated. The type of DNA replication initiated from these origins, termed constitutive stable DNA replication, does not require DnaA protein or the oriC site, which are essential for normal DNA replication. It requires active RecA protein. We previously found that the lexA71(Def)::Tn5 mutation can suppress this RecA requirement and postulated that the derepression of a LexA regulon gene(s) leads to the activation of a bypass pathway, Rip (for RecA-independent process). In this study, we isolated a miniTn10spc insertion mutant that abolishes the ability of the lexA(Def) mutation to suppress the RecA requirement of constitutive stable DNA replication. Cloning and DNA sequencing analysis of the mutant revealed that the insertion occurs at the 3' end of the coding region of the polA gene, which encodes DNA polymerase I. The mutant allele, designated polA25::miniTn10spc, is expected to abolish the polymerization activity but not the 5'-->3' or 3'-->5' exonuclease activity. Thus, the Rip bypass pathway requires active DNA polymerase I. Since the lethal combination of recA(Def) and polA25::miniTn10spc could be suppressed by derepression of the LexA regulon only when DNA replication is driven by the oriC system, it was suggested that the bypass pathway has a specific requirement for DNA polymerase I at the initiation step in the absence of RecA. An accompanying paper (Y. Cao and T. Kogoma, J. Bacteriol. 175:7254-7259, 1993) describes experiments to determine which activities of DNA polymerase I are required at the initiation step and discusses possible roles for DNA polymerase in the Rip bypass pathway.     

Perturbed chromosomal replication in recA mutants of Escherichia coli.

When initiation of DNA replication is inhibited in wild-type Escherichia coli cells by rifampin or chloramphenicol, completion of ongoing rounds of replication (runout of replication) leads to cells containing two, four, or eight fully replicated chromosomes, as measured by flow cytometry. In recombination-deficient recA strains, a high frequency of cells with three, five, six, or seven fully replicated chromosomes was observed in addition to cells with two, four, or eight chromosomes. recA mutants affected only in the protease-stimulating function behaved like wild-type cells. Thus, in the absence of the recombinase function of RecA protein, the frequency of productive initiations was significantly reduced compared with that in its presence. DNA degradation during runout of replication in the presence of rifampin was about 15%. The DNA degradation necessary to account for the whole effect described above was in this range or even lower. However, a model involving selective and complete degradation of partially replicated chromosomes is considered unlikely. It is suggested that the lack of RecA protein causes initiations or newly formed replication forks to stall but remain reactivatable for a period of time by functional RecA protein.