Specific cleavage of the p15A primer precursor by ribonuclease H at the origin of DNA replication.

We report studies on the mechanism of initiation of DNA replication by p15A, a small plasmid whose origin of replication is known to function much as does that of ColE1. Previous work has shown that an RNA primer for DNA synthesis is generated by the action of RNase H (EC 3.1.26.4) on a precursor transcript. The precursor initiates well upstream of the origin of replication and somehow forms a hybrid with its template during transcription. Here we show that when RNase H cleaves the hybrid at 0 degrees C, an additional cleavage product besides the primer can be identified. Using two-dimensional RNA sequencing techniques, we have established the sequence of this product to within a few nucleotides of each end. The position of the 5' end indicates that the nuclease introduces a nick or very small gap in the precursor at the origin. This suggests that some sequence or structure directs the enzyme to the origin. The position of its 3' end indicates that the precursor terminates at or near a series of six dAs in the template strand about 190 nucleotides from the origin of replication. The data indicate that hybrid formation may be necessary for termination of the precursor at this downstream site.

The importance of RNA secondary structure in CoIE1 primer formation.

Formation of the RNA primer for CoIE1 DNA replication is inhibited by random substitution of less than one tenth of G residues by I residues during in vitro transcription. Substitution in any one of several regions of the transcript is inhibitory, even in the region more than 400 nucleotides upstream of the origin of DNA replication. The inhibition results from interference with hybrid formation between nascent RNA II (primer transcript) and the template DNA near the replication origin. Association of RNA I with RNA II, which has been known to inhibit primer formation, enhances pausing of transcription of RNA II at a site far downstream of the region where RNA I hybridizes to the transcript. A large deletion in the region which specifies both RNA I and RNA II suppresses primer formation and also enhances pausing of transcription at the same site. These results show that the secondary structure of RNA II during transcription is important for primer formation and that alteration in the structure of the nascent transcript can change transcriptional events far downstream.

Detection of 5-methylcytosine in DNA sequences.

Col E1 DNA has methylated cytosine in the sequence 5'-CC*(A/T)GG-3' and methylated adenine in the sequence 5'-GA*TC-3' at the positions indicated by asterisks(*). When the Maxam-Gilbert DNA sequencing method is applied to this DNA, the methylated cytosine (5-methylcytosine) is found to be less reactive to hydrazine than are cytosine and thymine, so that a band corresponding to that base does not appear in the pyrimidine cleavage patterns. The existence of the methylated cytosine can be confirmed by analyzing the complementary strand or unmethylated DNA. In contrast, the methylated adenine (probably N6-methyladenine) cannot be distinguished from adenine with standard conditions for cleavage at adenine.

Nalidixic acid resistance: a second genetic character involved in DNA gyrase activity.

ATP-dependent DNA supercoiling catalyzed by Escherichia coli DNA gyrase was inhibited by oxolinic acid, a compound similar to but more potent than nalidixic acid and a known inhibitor of DNA replication in E. coli. The supercoiling activity of DNA gyrase purified from nalidixic acid-resistant mutant (nalA(R)) bacteria was resistant to oxolinic acid. Thus, the nalA locus is responsible for a second component needed for DNA gyrase activity in addition to the component determined by the previously described locus for resistance to novobiocin and coumermycin (cou). Supercoiling of lambda DNA in E. coli cells was likewise inhibited by oxolinic acid, but was resistant in the nalA(R) mutant. The inhibition by oxolinic acid of colicin E1 plasmid DNA synthesis in a cell-free system was largely relieved by adding resistant DNA gyrase. In the absence of ATP, DNA gyrase preparations relaxed supercoiled DNA; this activity was also inhibited by oxolinic acid, but not by novobiocin. It appears that the oxolinic acid-sensitive component of DNA gyrase is involved in the nicking-closing activity required in the supercoiling reaction. In the presence of oxolinic acid, DNA gyrase forms a complex with DNA, which can be activated by later treatment with sodium dodecyl sulfate and a protease to produce double-strand breaks in the DNA. This process has some similarities to the known properties of relaxation complexes.

Origin of replication of colicin E1 plasmid DNA.

Cleavage maps of colicin E1 plasmid DNA and its smaller derivative, pNT1 DNA, were constructed by using restriction endonucleases. The nucleotide sequence of a region that contains the orgin of replication was determined. The site of the nucleotide from which DNA replication is initiated was determined with 6S L-fragments, the DNA fragment first made on colicin E1 plasmid DNA. The fragments were labeled with [gamma-32P]ATP and polynucleotide 5'-hydroxyl-kinase (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) at the 5'-OH groups which were uncovered by alkali treatment. The site is one of three consecutive nucleotides, dA, dA, and dC, located at a unique position. One or a few rA residues were found to be attached to some of the DNA molecules. The transition from the primer RNA to DNA occurs in a region consisting of a segment of five A residues. Both sides of this segment are rich in G and C.

Replication of colicin E1 plasmid DNA added to cell extracts.

Closed-circular DNA of colicin E1 plasmid can undergo a round of semiconservative replication when added to an extract of Escherichia coli. Extracts of cells that do not carry the plasmid are able to perform complete replication of the plasmid. Replication requires de novo RNA synthesis but not protein synthesis.

Termination point of replication of colicin E1 plasmid DNA in cell extracts.

Closed-circular monomeric molecules were one of the major products of replication of colicin E1 plasmid DNA in cell extracts. However, when the plasmid DNA synthesized in the reaction mixture was labeled for 3 min after 27 min of incubation, most of the label was found in open-circular molecules. The open-circular molecules were converted to closed-circular molecules upon further incubation. The newly replicated open-circular molecules had a nick or small gap in their newly synthesized strand. The interruption was located at approximately 20% of the molecular length from the single site of cleavage by restriction endonuclease EcoR1 and at or very close to the origin of replication. The addition of 10 mM nicotinamide mononucleotide instead of nicotinamide adenine dinucleotide to extracts did not significantly affect the kinetics of the colicin E1 plasmid DNA synthesis. However, in the presence of nicotinamide mononucleotide the formation of completely replicated closed-circular molecules was suppressed and, instead, open-circular molecules accumulated with an interruption in the newly synthesized strand at the termination point of replication, which was located at or very close to the origin of replication.

Replication of colicin E1 plasmid DNA in cell extracts.

Cell extracts were prepared from Escherichia coli carrying colicin E1 plasmid. The DNA in extracts was almost exclusively closed-circular DNA of the plasmid. Labeled deoxyribonucleotides were incorporated into DNA in extracts. DNA of colicin E1 plasmid was the sole DNA product, and was composed of completely replicated molecules and a class of replicative intermediates. The intermediates carried an average of approximately two pieces of DNA fragments that had a sedimentation coefficient of approximately 6 S and were not covalently attached to the parental DNA strands. The replication was initiated on closed-circular molecules and the complete molecules were synthesized semiconservatively. The DNA synthesis depended on the four ribonucleoside triphosphates and was sensitive to rifampicin. A round of DNA replication, once initiated, was completed in the presence of rifampicin, indicating that RNA synthesis is involved in the initiation of replication of the plasmid DNA. Most of the replicated molecules were isolated in super-coiled structures. These results indicate that this soluble system is capable of carrying out a complete round of replication of colicin E1 plasmid DNA.