RNase HI overproduction is required for efficient full-length RNA synthesis in the absence of topoisomerase I in Escherichia coli

It has long been known that Escherichia coli cells deprived of topoisomerase I (topA null mutants) do not grow. Because mutations reducing DNA gyrase activity and, as a consequence, negative supercoiling, occur to compensate for the loss of topA function, it has been assumed that excessive negative supercoiling is somehow involved in the growth inhibition of topA null mutants. However, how excess negative supercoiling inhibits growth is still unknown. We have previously shown that the overproduction of RNase HI, an enzyme that degrades the RNA portion of an R-loop, can partially compensate for the growth defects because of the absence of topoisomerase I. In this article, we have studied the effects of gyrase reactivation on the physiology of actively growing topA null cells. We found that growth immediately and almost completely ceases upon gyrase reactivation, unless RNase HI is overproduced. Northern blot analysis shows that the cells have a significantly reduced ability to accumulate full-length mRNAs when RNase HI is not overproduced. Interestingly, similar phenotypes, although less severe, are also seen when bacterial cells lacking RNase HI activity are grown and treated in the same way. All together, our results suggest that excess negative supercoiling promotes the formation of R-loops, which, in turn, inhibit RNA synthesis.     

Function of RNase H in DNA replication revealed by RNase H defective mutants of Escherichia coli

Summary Escherichia coli rnh mutants were isolated using localized mutagenesis and selective measurements of RNase H activity in mutagenized cell extracts with [³H]poly(rC)·poly(dG) as substrate. RNase H activity in extracts of one mutant, ON152 (rnh-91), was undetectable (less than 0.05% of that of wild-type cells). This mutant formed small colonies at 43 °C. At this temperature, accumulation of nascent fragments was more prominent in the rnh-91·polA4113 double mutant than in the polA4113 mutant; however, no accumulation was found in the rnh single mutant at 43° C. Unlike the 1–3 nucleotide primer RNA found on nascent fragments of polA4113 cells, primers from the rnh-91·polA4113 cells ranged from one to about ten bases. These results suggest that the 53 exonuclease activity of DNA polymerase I plays a major role in removal of primer RNA and that RNase H functions in an auxiliary role, excising the 5-portion of longer primers.The rnh mutant supports replication of ColE1-type plasmids. A possible mechanism of replication of such plasmids in rnh mutants and a role of RNase H in the initiation of chromosomal replication are discussed.     

RNase H and replication of ColE1 DNA in Escherichia coli

Amber mutations within the rnh (RNase H) gene of Escherichia coli K-12 were isolated by selecting for bacteria capable of replicating in a sup+ background replication-defective cer-6 mutant of the ColE1 replicon. The cer-6 mutation is an alteration of one base pair located 160 nucleotides upstream of the unique replication origin of this plasmid. Subsequently, we determined the DNA alterations present within these mutants. ColE1 DNA replicated in rnh(Am) recA cells, indicating that (i) RNase H, which has been shown to be absolutely required for in vitro initiation of ColE1 DNA replication, is dispensable in vivo, and (ii) ColE1 replication in the absence of RNase H is not dependent on "stable DNA replication," which has been reported to be an alternative mode of chromosomal DNA replication. Another class of bacterial mutations was also isolated. These mutations, named herB, suppressed cer-6 replication in rnh+ bacteria. herB mutations mapped close to the polA gene on the E. coli chromosome and increased the activity of DNA polymerase I. These findings suggest that when the DNA polymerase I has an opportunity to initiate DNA synthesis before RNase H acts, the replication-defective cer-6 mutant or the wild-type ColE1 replicates in E. coli.     

Viability of Escherichia coli topA mutants lacking DNA topoisomerase I

The viability of the topA mutants lacking DNA topoisomerase I was thought to depend on the presence of compensatory mutations in Escherichia coli but not Salmonella typhimurium or Shigella flexneri. This apparent discrepancy in topA requirements in different bacteria prompted us to reexamine the topA requirements in E. coli. We find that E. coli strains bearing topA mutations, introduced into the strains by DNA-mediated gene replacement, are viable at 37 or 42 degrees C without any compensatory mutations. These topA(-) cells exhibit cold sensitivity in their growth, however, and this cold sensitivity phenotype appears to be caused by excessive negative supercoiling of intracellular DNA. In agreement with previous results (Zhu, Q., Pongpech, P., and DiGate, R. J. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 9766-9771), E. coli cells lacking both type IA DNA topoisomerases I and III are found to be nonviable, indicating that the two type IA enzymes share a critical cellular function.     

Characterization of vaccinia virus DNA topoisomerase I expressed in Escherichia coli

The putative structural gene encoding the vaccinia virus type I DNA topoisomerase (EC 5.99.1.2) was expressed in Escherichia coli under the control of a bacteriophage T7 promoter. Provision of T7 RNA polymerase resulted in the accumulation to high level of a Mr = 33,000 type I topoisomerase with the properties of the vaccinia enzyme. A simple purification scheme yielded approximately 8 mg of recombinant vaccinia topoisomerase from 400 ml of bacteria. DNA unwinding by the enzyme was stimulated by magnesium, manganese, calcium, cobalt, and spermidine, but inhibited by copper and zinc. Like eukaryotic cellular type I topoisomerases, but unlike the prokaryotic counterpart, the recombinant topoisomerase relaxed positively and negatively supercoiled DNA. The viral topoisomerase I was, however, resistant to the effects of camptothecin, a drug that specifically inhibits cellular type I topoisomerases.     

Analysis of mRNA decay and rRNA processing in Escherichia coli in the absence of RNase E-based degradosome assembly

We demonstrate here that the assembly of the RNase E-based degradosome of Escherichia coli is not required for normal mRNA decay in vivo. In contrast, deletion of the arginine-rich RNA binding site (ARRBS) from the RNase E protein slightly impairs mRNA decay. When both the degradosome scaffold region and the ARRBS are missing, mRNA decay is dramatically slowed, but 9S rRNA processing is almost normal. An extensive RNase E truncation mutation (rnedelta610) had a more pronounced mRNA decay defect at 37 degrees C than the temperature-sensitive rne-1 allele at 44 degrees C. Taken together, these data suggest that the inviability associated with inactivation of RNase E is not related to defects in either mRNA decay or rRNA processing.     

Genetic inactivation of topoisomerase I suppresses a defect in initiation of chromosome replication in Escherichia coli

Summary A strain of Escherichia coli K12 harboring simultaneously the temperature-sensitive dnaA46 mutation and a deletion of the trp-topA-cysB region plates with the same full efficiency at 30° C and 42° C. We have analyzed the possible involvement of the gene coding for topoisomerase I, topA, in this suppression phenomenon. The Ts phenotype was retrieved upon introduction of a plasmid-borne DNA fragment including an active topA gene into this strain, but not upon introduction of the same fragment harboring a topA::Tn1000 insertion. Replication seems to remain DnaA-dependent in the (topA) strain, however, since we have been unable to introduce a dnaA::Tn10 allele. We propose either that the dnaA46 gene product is overproduced and compensates for its thermal inactivation, or that initiation at oriC demands less DnaA protein in the absence of topoisomerase I.Abbreviations Ap^r ampicillin resistance - Cm^r chloramphenicol resistance - Km^r Kanamycin resistance - St^r streptomycin resistance - Tc^r tetracycline resistance     

Effects of modulation of RNase H production on the recovery of DNA synthesis following UV-irradiation in Escherichia coli

The requirements for the recovery of DNA synthesis in UV-irradiated Escherichia coli were analysed in strains having varied levels of RNase H and RecA protein. We have previously shown (Khidhir et al. 1985) that the recovery of DNA synthesis in E. coli following UV treatment is an inducible SOS function requiring protein synthesis. We proposed that this reflected the need for the synthesis of specific induced replisome reactivation factor(s) for recovery. In this study we now show that recovery of DNA synthesis can in fact take place in the absence of protein synthesis in a mutant lacking RNase H and having high (constitutive) levels of RecA protein. We also show that expression of rnh is inhibited during the SOS response in recA+ but not in a recA- strain. The results are discussed in relation to the mechanism of recovery of DNA synthesis following UV irradiation in E. coli.     

Escherichia coli DNA topoisomerase I mutants have compensatory mutations in DNA gyrase genes

Escherichia coli deletion mutants lacking DNA topoisomerase I have been identified previously and shown to grow at a normal rate. We show that such strains grow normally only because of spontaneously arising mutations that compensate for the topoisomerase I defect. Several of these compensatory mutations have been found to map at or near the genes encoding DNA gyrase, gyrA and gyrB. DNA gyrase assays of crude extracts show that strains carrying the mutations have lower gyrase activity. Thus the mutations are in the gyrase structural genes or in nearby regulatory sequences. These results, in conjunction with DNA supercoiling measurements of others, indicate that in vivo DNA superhelicity is a result of a balance between topoisomerase I and gyrase activities. An excess of negative supercoils due to an absence of topoisomerase I is deleterious to the cell, but a moderate gyrase deficiency is not harmful.     

Effect of phosphorothioate substitutions on DNA cleavage by Escherichia coli DNA topoisomerase I

To evaluate the structural influence of the DNA phosphate backbone on the activity of Escherichia coli DNA topoisomerase I, modified forms of oligonucleotide dA(7) were synthesized with a chiral phosphorothioate replacing the non-bridging oxygens at each position along the backbone. A deoxy-iodo-uracil replaced the 5'-base to crosslink the oligonucleotides by ultraviolet (UV) and assess binding affinity. At the scissile phosphate there was little effect on the cleavage rate. At the +1 phosphate, the rectus phosphorus (Rp)-thio-substitution reduced the rate of cleavage by a factor of 10. At the +3 and -2 positions from the scissile bond, the Rp-isomer was cleaved at a faster rate than the sinister phosphorus (Sp)-isomer. The results demonstrate the importance of backbone contacts between DNA substrate and E. coli topoisomerase I.     

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     

RNase I*, a form of RNase I, and mRNA degradation in Escherichia coli.

A previously unreported endoRNase present in the spheroplast fraction of Escherichia coli degraded homoribopolymers and small RNA oligonucleotides but not polymer RNA. Like the periplasmic endoRNase, RNase I, the enzyme cleaved the phosphodiester bond between any nucleotides; however, RNase I degraded polymer RNA as fast as homopolymers or oligomers. Both enzymes migrated as 27-kDa polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and could not be separated by various chromatographic procedures. In rna insertion mutants, both enzymes were completely missing; the spheroplast enzyme is called RNase I*, since it must be a form of RNase I. The two forms could be distinguished by physical treatments. RNase I could be activated by Zn2+, while RNase I* was inactive in the presence of Zn2+. RNase I was inactivated very slowly at 100 degrees C over a wide pH range, while RNase I* was inactivated slowly by heat at pH 4.0 but much more rapidly as the pH was increased to 8.0. In the presence of a thiol-binding agent, the inactivation at the higher pH values was much slower. These results suggest that RNase I*, but not RNase I, has free sulfhydryl groups. RNase I* activity in the cell against a common substrate was estimated to be several times that of RNase I. All four 2',3'-phosphomonoribonucleotides were identified in the soluble pools of growing cells. Such degradative products must arise from RNase I* activity. The activity would be suited for the terminal step in mRNA degradation, the elimination of the final oligonucleotide fragments, without jeopardizing the cell RNA. An enzyme with very similar specificity was found in Saccharomyces cerevisiae, suggesting that the activity may be widespread in nature.     

Flexibility at Gly-194 is required for DNA cleavage and relaxation activity of Escherichia coli DNA topoisomerase I

The proposed mechanism of type IA DNA topoisomerase I includes conformational changes by the single enzyme polypeptide to allow binding of the G strand of the DNA substrate at the active site, and the opening or closing of the "gate" created on the G strand of DNA to the passing single or double DNA strand(s) through the cleaved G strand DNA. The shifting of an alpha helix upon G strand DNA binding has been observed from the comparison of the type IA DNA topoisomerase crystal structures. Site-directed mutagenesis of the strictly conserved Gly-194 at the N terminus of this alpha helix in Escherichia coli DNA topoisomerase I showed that flexibility around this glycine residue is required for DNA cleavage and relaxation activity and supports a functional role for this hinge region in the enzyme conformational change.     

Escherichia coli RNase M is a multiply altered form of RNase I.

RNase M, an enzyme previously purified to homogeneity from Escherichia coli, was suggested to be the RNase responsible for mRNA degradation in this bacterium. Although related to the endoribonuclease, RNase I, its distinct properties led to the conclusion that RNase M was a second, low molecular mass, broad specificity endoribonuclease present in E. coli. However, based on sequence analysis, southern hybridization, and enzyme activity, we show that RNase M is, in fact, a multiply altered form of RNase I. In addition to three amino acid substitutions that confer the properties of RNase M on the mutated RNase I, the protein is synthesized from an rna gene that contains a UGA nonsense codon at position 5, apparently as a result of a low level of readthrough. We also suggest that RNase M is just one of several previously described endoribonuclease activities that are actually manifestations of RNase I.