RNase H-defective mutants of Escherichia coli: a possible discriminatory role of RNase H in initiation of DNA replication

Summary Mutants of Escherichia coli completely deficient in RNase H activity were isolated by inserting transposon Tn3 into the structural gene for RNase H, rnh, and its promoter. These rnh ^- mutants exhibited the following phenotypes; (1) the mutants grew fairly normally, (2) rnh ^- cells could be transformed with ColE1 derivative plasmids, pBR322 and pML21, though the plasmids were relatively unstable, under non selective conditions, (3) rnh ^- mutations partially suppressed the temperature-sensitive phenotype of plasmid pSC301, a DNA replication initiation mutant derived from pSC101, (4) rnh ^- mutations suppressed the temperature-sensitive growth character of dnaA ^ts mutant, (5) rnh ^- cells showed continued DNA synthesis in the presence of chloramphenicol (stable DNA replication). Based on these findings we propose a model for a role of RNase H in the initiation of chromosomal DNA replication. We suggest that two types of RNA primers for initiation of DNA replication are synthesized in a dnaA/oriC-dependent and-independent manner and that only the dnaA/oriC-dependent primer is involved in the normal DNA replication since the dnaA/oriC independent primer is selectively degraded by RNase H.Abbreviations AP^r ampicillin-resistant - kb kilobase pair(s) - NEM N-ethyl maleimide - Ts temperature-sensitive     

RecA protein acts at the initiation of stable DNA replication in rnh mutants of Escherichia coli K-12.

Escherichia coli rnh mutants lacking RNase H activity are capable of recA+-dependent DNA replication in the absence of concomitant protein synthesis (stable DNA replication). In rnh dnaA::Tn10 and rnh delta oriC double mutants in which the dnaA+-dependent initiation of DNA replication at oriC is completely blocked, the recA200 mutation encoding a thermolabile RecA protein renders both colony formation and DNA synthesis of these mutants temperature sensitive. To determine which stage of DNA replication (initiation, elongation, or termination) was blocked, we analyzed populations of these mutant cells incubated at 30 or 42 degrees C in the presence or absence of chloramphenicol (CM) by dual-parameter (DNA-light scatter) flow cytometry. Incubation at 30 degrees C in the presence of CM resulted in cells with a continuum of DNA content up to seven or more chromosome equivalents per cell. The cultures which had been incubated at 42 degrees C in the absence or presence of CM consisted of cells with integral numbers of chromosomes per cell. It is concluded that active RecA protein is required specifically for the initiation of stable DNA replication.     

Genetic analysis of constitutive stable DNA replication in rnh mutants of Escherichia coli K12

Summary Escherichia coli rnh mutants deficient in ribonuclease H (RNase H) are capable of DNA replication in the absence of protein synthesis. This constitutive stable DNA replication (SDR) is dependent upon the recA ⁺ gene product. The requirement of SDR for recA ⁺ can be suppressed by rin mutations (for recA⁺-independent), or by lexA(Def) mutations which inactivate the LexA repressor. Thus, there are at least three genetically distinct types of SDR in rnh mutants: recA ⁺-dependent SDR seen in rnh ^- rin⁺ lexA⁺ strains, recA ⁺-independent in rnh ^- rin^- lexA⁺, and recA ⁺-independent in rnh ^- rin⁺ lexA(Def). The expression of SDR in rin ^- and lexA(Def) mutants demonstrated a requirement for RNA synthesis and for the absence of RNase H. The suppression of the recA ⁺ requirement by rin mutations was shown to depend on some new function of the recF ⁺ gene product. In contrast, the suppression by lexA-(Def) mutations was not dependent on recF ⁺. The lexA3 mutation inhibited recA ⁺-dependent SDR via reducing the amount of recA ⁺ activity available, and was suppressed by the recAo254 mutation. The SDR in rnh ^- rin^- cells was also inhibited by the lexA3 mutation, but the inhibition was not reversed by the recAo254 mutation, indicating a requirement for some other lexA ⁺-regulated gene product in the recA ⁺-independent SDR process. A model is presented for the regulation of the expression of these three types of SDR by the products of the lexA ⁺, rin⁺ and recF ⁺ genes.     

DNA polymerase I in constitutive stable DNA replication in Escherichia coli.

We examined the effects of mutations in the polA (encoding DNA polymerase I) and polB (DNA polymerase II) genes on inducible and constitutive stable DNA replication (iSDR and cSDR, respectively), the two alternative DNA replication systems of Escherichia coli. The polA25::miniTn10spc mutation severely inactivated cSDR, whereas polA1 mutants exhibited a significant extent of cSDR. cSDR required both the polymerase and 5'-->3' exonuclease activities of DNA polymerase I. A similar requirement for both activities was found in replication of the pBR322 plasmid in vivo. DNA polymerase II was required neither for cSDR nor for iSDR. In addition, we found that the lethal combination of an rnhA (RNase HI) and a polA mutation could be suppressed by the lexA(Def) mutation.     

Hyper-recombination in Escherichia coli K-12 mutants constitutive for protein X synthesis.

Genetic recombination was studied in Escherichia coli F- strains in which synthesis of the recA gene product protein X is increased due to mutation in either recA (tif-1) or lexA (spr). When a single donor marker was selected, the recombination proficiency of these strains was not significantly altered in Hfr crosses. However, linkage of unselected, proximal Hfr markers was found to be much reduced among the progeny tested, and more of the progeny showed evidence of multiple exchanges between donor and recipient DNA. These effects were much more apparent when the recipient carried both tif-1 and spr mutations, but in this case recombination proficiency was reduced compared with those strains carrying either mutation alone, particularly in crosses with Hfr Cavalli. A lexA mutation was found to suppress the effect of tif-1 on the recombinant genotype.     

The initiation mass for DNA replication in Escherichia coli K-12 is dependent on growth rate.

It is widely accepted that the initiation mass of Escherichia coli is constant and independent of growth rate, and therefore is an important parameter in the regulation of initiation of DNA replication. We have used flow cytometry to measure the initiation mass of E. coli K-12 cells as a function of growth rate. The average initiation mass was determined by two methods: (i) from a mathematical relationship between average cell mass, cell age at initiation and number of origins present in the cells, and (ii) directly from the cell mass distribution. The light scattering signal from individual cells and the protein content per cell were employed as measures of cell mass. The initiation mass was found to increase monotonically with decreasing growth rate, being 1.6 times higher (light scattering) or 2.1 times higher (protein content) at 0.3 than at 2.5 doublings per hour. We conclude that the initiation mass is dependent on growth rate. This finding indicates that the control for timing of initiation is not governed by a direct connection between mass accumulation and the molecule(s) determining initiation of 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.     

Nonrandom F-plasmid replication in Escherichia coli K-12.

Both the autonomous and chromosomally integrated F plasmids were found to replicate in a nonrandom fashion after a density transfer from heavy medium ([13C]glucose, 15NH4) to light medium ([12C]glucose, 14NH4). The data are consistent with the hypothesis that both the chromosome and the F plasmid are replicated in a cell cycle-specific manner. Thus, these data support the proposal (J. D. Keasling, B. O. Palsson, and S. Cooper, J. Bacteriol. 173:2673-2680, 1991) that plasmids replicate in a cell cycle-specific manner.     

Nonrandom minichromosome replication in Escherichia coli K-12.

The intervals between rounds of chromosome and minichromosome replication were measured by density shift experiments and found to be similar. Thus the minichromosome, a lambda asnA oriC bacteriophage, mostly replicates once each division cycle rather than randomly, despite its high copy number. Slight differences between the chromosome and the oriC plasmid are explained.     

Characteristics of cold-sensitive mutants of Escherichia coli K-12 defective in deoxyribonucleic acid replication.

Four cold-sensitive mutants of Escherichia coli have been isolated which show a reduced ability to synthesize deoxyribonucleic acid at low temperature. The mutants also have a reduced ability to incorporate nucleoside triphosphates into deoxyribonucleic acid at low temperature in cell preparations made permeable with toluene. All four mutations are located at or near the dnaA locus on the E. coli genetic map. They are recessive to the wild-type allele and two of them can be integratively suppressed by F episomes.     

Potassium-dependant mutants of Escherichia coli K-12

Mutants of Escherichia coli K-12 that grow more slowly in media containing low concentrations of K have been isolated. All independent mutants of this type which have been studied carry a mutation in a small region of the bacterial chromosome between the supE and gal loci. The growth rate of the mutants is the same as that of the parental strains in medium containing more than 1 mm K, but is only 50% that of the parent when the K concentration is reduced to 0.1 mm. The mutants do not appear to have a primary alteration in K transport, and are therefore referred to as K-dependent. The abbreviation kdp is proposed for this class of mutant.     

Flavodoxin mutants of Escherichia coli K-12

The flavodoxins are flavin mononucleotide-containing electron transferases. Flavodoxin I has been presumed to be the only flavodoxin of Escherichia coli, and its gene, fldA, is known to belong to the soxRS (superoxide response) oxidative stress regulon. An insertion mutation of fldA was constructed and was lethal under both aerobic and anaerobic conditions; only cells that also had an intact (fldA(+)) allele could carry it. A second flavodoxin, flavodoxin II, was postulated, based on the sequence of its gene, fldB. Unlike the fldA mutant, an fldB insertion mutant is a viable prototroph in the presence or absence of oxygen. A high-copy-number fldB(+) plasmid did not complement the fldA mutation. Therefore, there must be a vital function for which FldB cannot substitute for flavodoxin I. An fldB-lacZ fusion was not induced by H(2)O(2) and is therefore not a member of the oxyR regulon. However, it displayed a soxS-dependent induction by paraquat (methyl viologen), and the fldB gene is preceded by two overlapping regions that resemble known soxS binding sites. The fldB insertion mutant did not have an increased sensitivity to the effects of paraquat on either cellular viability or the expression of a soxS-lacZ fusion. Therefore, fldB is a new member of the soxRS (superoxide response) regulon, a group of genes that is induced primarily by univalent oxidants and redox cycling compounds. However, the reactions in which flavodoxin II participates and its role during oxidative stress are unknown.     

L-Serine-sensitive mutants of Escherichia coli K-12

While attempting to isolate d-serine-sensitive mutants of Escherichia coli K-12, we found a class of mutants sensitive to low concentrations of l-serine (10 to 25 mug/ml).