Identification of a primosome assembly site in the region of the ori 2 replication origin of the Escherichia coli mini-F plasmid.

A primosome assembly site for F plasmid DNA replication has been identified. This site, which we term rriA (F), is localized to one strand of a 385-base-pair Sau3A restriction fragment very close to ori 2 and within the 2.25-kilobase DNA sequence required for replication and incompatibility of the entire F plasmid. rriA (F) was isolated by cloning into the deletion phage vector M13 delta Elac. This phage forms very faint plaques due to a deletion of the M13 complementary strand origin but forms large wild-type plaques when DNA single-strand initiation determinants are inserted. The single-stranded viral DNA of the Sau3A F-M13 delta Elac recombinant provides an effector site of dATP hydrolysis by the primosomal protein n'. It also provides an assembly site for the Escherichia coli primosome protein complex that directs the in vitro conversion of the single-stranded DNA to a double-stranded form by the same mechanism as that used by phi X174. Homologies of the nucleotide sequence between this F DNA sequence and the previously identified primosome assembly sites in phi X174 phage DNA and in ColE1 plasmid DNA (rriA and rriB) have been found. The sequences 5' G-T-G-A-G-C-G 3' and 5' G-N-G-G-A-A-G-C 3' or variations of these sequences occur from two to five times within each assembly locus. In addition, two distinct 15-base-pair sequences in rriA (F) are perfectly homologous to corresponding sequences in rriA (ColE1).     

Structure of the ColE1 DNA molecule before segregation to daughter molecules.

The segregation of daughter DNA molecules at the end stage of replication of plasmid ColE1 was examined. When circular ColE1 DNA replicates in a cell extract at a high KCl concentration (140 mM), a unique class of molecules accumulates. When the molecule is cleaved by a restriction enzyme that cuts the ColE1 DNA at a single site, an X-shaped molecule in which two linear components are held together around the origin of DNA replication is made. For a large fraction of these molecules, the 5' end of the leading strand remains at the origin and the 3' end of the strand is about 30 nucleotides upstream of the origin. The 3' end of the lagging strand is located at the terH site (17 nucleotides upstream of the origin) and the 5' end of the strand is a few hundred nucleotides upstream of the terH site. Thus the parental strands of the molecule intertwine with each other only once. When the KCl concentration is lowered to 70 mM, practically all of these molecules are converted to daughter circular monomers or to catenanes consisting of two singly interlocked circular units.     

Escherichia coli mutants suppressing replication-defective mutations of the ColE1 plasmid.

Mutants of Escherichia coli K-12 have been isolated that suppress cer mutants, ColE1 mutants that are unable to replicate as the plasmid. These host suppressors were designated her, for host factor affecting ColE1 replication. Each her suppressor showed a characteristic pattern of suppression depending on the cer mutation used for selecting the mutant bacteria. One of the suppressors, named herA, that suppressed cer6, a single-base-pair alteration 160 base pairs upstream of the ColE1 replication origin, was genetically identified as an alteration of the rnh gene (RNase H). HerA was recessive to its wild-type allele. RNase H activity of herA cell extracts was defective. Conversely, rnh mutants that were isolated independently of ColE1 replication supported replication of cer6 DNA. Some rnh mutants manifested the HerA phenotype only above a certain transition temperature, and their RNase H activity was found to be temperature sensitive. Therefore, replication of cer6 DNA in vivo is sensitive to RNase H activity. Under the conditions that suppressed cer6, the wild-type colE1 replicon replicated normally. Then, ColE1 replication in vivo proceeds in the absence of RNase H activity, which has been shown to be required for in vitro replication of the DNA.     

Isolation of Supercoiled Colicinogenic Factor E1 DNA Sensitive to Ribonuclease and Alkali

The synthesis of the covalently-closed, circular DNA form of colicinogenic factor E(1) (ColE(1)) continues in Escherichia coli cells after the addition of chloramphenicol. A large portion of the purified supercoiled ColE(1) DNA molecules made in the presence of chloramphenicol are converted to the open circular DNA form after treatment with alkali (pH 13), RNase A, or RNase H. These treatments do not significantly affect the covalently-closed form of ColE(1) DNA isolated from normally growing E. coli cells. The open circular product resulting from treatment of supercoiled ColE(1) DNA with RNase A possesses a single break in one strand of the circular duplex. The site sensitive to RNase A occurs with equal probability in either of the complementary strands. Both synthesis of ColE(1) DNA and the formation of supercoiled ColE(1) DNA sensitive to RNase A or alkali are prevented by the inhibitor of RNA synthesis, rifampicin. These results indicate that covalently-closed ColE(1) DNA containing one or more ribonucleotides accumulates during ColE(1) replication in the presence of chloramphenicol. It is proposed that this incorporated RNA served as a primer during the initiation of synthesis of ColE(1) DNA and that its removal from the circular DNA is inhibited in cells incubated in the presence of chloramphenicol.     

Enzymatic completion of mammalian lagging-strand DNA replication.

Using purified proteins from calf and a synthetic substrate, we have reconstituted the enzymatic reactions required for mammalian Okazaki fragment processing in vitro. The required reactions are removal of initiator RNA, synthesis from an upstream fragment to generate a nick, and then ligation. With our substrate, RNase H type I (RNase HI) makes a single cut in the initiator RNA, one nucleotide 5' of the RNA-DNA junction. The double strand specific 5' to 3' exonuclease removes the remaining monoribonucleotide. After dissociation of cleaved RNA, synthesis by DNA polymerase generates a nick, which is then sealed by DNA ligase I. The unique specificities of the two nucleases for primers with initiator RNA strongly suggest that they perform the same reactions in vivo.