Primer RNA synthesis by plasmid-specified Rep protein for initiation of ColE2 DNA replication.

Initiation of in vitro ColE2 DNA replication requires the plasmid-specified Rep protein and DNA polymerase I but not RNA polymerase and DnaG primase. The ColE2 Rep protein binds specifically to the origin where replication initiates. Leading-strand synthesis initiates at a unique site in the origin and lagging-strand DNA synthesis terminates at another unique site in the origin. Here we show that the primer RNA for leading-strand synthesis at the origin has a unique structure of 5'-ppApGpA. We reconstituted the initiation reaction of leading-strand DNA synthesis by using purified proteins, the ColE2 Rep protein, Escherichia coli DNA polymerase I and SSB, and we showed that the ColE2 Rep protein is a priming enzyme, primase, which is specific for the ColE2 origin. The ColE2 Rep protein is unique among other primases in that it recognizes the origin region and synthesizes the primer RNA at a fixed site in the origin region. Specific requirement for ADP as a substrate and its direct incorporation into the 5' end of the primer RNA are also unique properties of the ColE2 Rep protein.     

Initiation site of deoxyribonucleotide polymerization at the replication origin of the Escherichia coli chromosome

A new round of chromosomal replication of a temperature-sensitive initiation mutant (dnaC) of Escherichia coli was initiated synchronously by a temperature shift from a nonpermissive to a permissive condition in the presence of arabinosyl cytosine. Increased amounts of nascent DNA fragments with homology for the chromosomal segment containing the replication origin (oriC) were found. The nascent DNA fragments were purified and treated with alkali to hydrolyze putative primer RNA and to expose 5'-hydroxyl DNA ends at the RNA-DNA junctions. The ends were then labeled selectively with T4 polynucleotide kinase and [gamma-32P]ATP at 0 degrees C and the terminally-labeled initiation fragments were purified by hybridization with origin probe DNAs containing one each of the constituent strands of oriC-DNA segment. The 32P-labeled initiation sites were then located at the resolution of single nucleotides in the nucleotide sequence of the oriC segment after cleavage with restriction enzymes. Two initiation sites of DNA synthesis, 37 nucleotides apart, were detected in one of the component strands of the oriC; in other words, in the strand whose 5' to 3' polynucleotide polarity lies counterclockwise on the E. coli genetic map. The results support the involvement of the primer RNA in the initiation of DNA synthesis at the origin of the E. coli genome and suggest that the first initiation event is asymmetric.     

Suppression of ColE1 high-copy-number mutants by mutations in the polA gene of Escherichia coli.

We isolated three Escherichia coli suppressor strains that reduce the copy number of a mutant ColE1 high-copy-number plasmid. These mutations lower the copy number of the mutant plasmid in vivo up to 15-fold; the wild-type plasmid copy number is reduced by two- to threefold. The suppressor strains do not affect the copy numbers of non-ColE1-type plasmids tested, suggesting that their effects are specific for ColE1-type plasmids. Two of the suppressor strains show ColE1 allele-specific suppression; i.e., certain plasmid copy number mutations are suppressed more efficiently than others, suggesting specificity in the interaction between the suppressor gene product and plasmid replication component(s). All of the mutations were genetically mapped to the chromosomal polA gene, which encodes DNA polymerase I. The suppressor mutational changes were identified by DNA sequencing and found to alter single nucleotides in the region encoding the Klenow fragment of DNA polymerase I. Two mutations map in the DNA-binding cleft of the polymerase region and are suggested to affect specific interactions of the enzyme with the replication primer RNA encoded by the plasmid. The third suppressor alters a residue in the 3'-5' exonuclease domain of the enzyme. Implications for the interaction of DNA polymerase I with the ColE1 primer RNA are discussed.     

Functional features of an ssi signal of plasmid pGKV21 in Escherichia coli.

A single-strand initiation (ssi) signal was detected on the Lactococcus lactis plasmid pGKV21 containing the replicon of pWV01 by its ability to complement the poor growth of an M13 phage derivative (M13 delta lac182) lacking the complementary-strand origin in Escherichia coli. This ssi signal was situated at the 229-nucleotide (nt) DdeI-DraI fragment and located within the 109 nt upstream of the nick site of the putative plus origin. SSI activity is orientation specific with respect to the direction of replication. We constructed an ssi signal-deleted plasmid and then examined the effects of the ssi signal on the conversion of the single-stranded replication intermediate to double-stranded plasmid DNA in E. coli. The plasmid lacking an ssi signal accumulated much more plasmid single-stranded DNA than the wild-type plasmid did. Moreover, deletion of this region caused a great reduction in plasmid copy number or plasmid maintenance. These results suggest that in E. coli, this ssi signal directs its lagging-strand synthesis as a minus origin of plasmid pGKV21. Primer RNA synthesis in vitro suggests that E. coli RNA polymerase directly recognizes the 229-nt ssi signal and synthesizes primer RNA dependent on the presence of E. coli single-stranded DNA binding (SSB) protein. This region contains two stem-loop structures, stem-loop I and stem-loop II. Deletion of stem-loop I portion results in loss of priming activity by E. coli RNA polymerase, suggesting that stem-loop I portion is essential for priming by E. coli RNA polymerase on the SSB-coated single-stranded DNA template.     

Discriminatory function of ribonuclease H in the selective initiation of plasmid DNA replication.

The initiation stage of ColE1-type plasmid replication was reconstituted with purified protein fractions from Escherichia coli. The reconstituted system included DNA polymerase I, DNA ligase, RNA polymerase, DNA gyrase, and a discriminating activity copurifying with RNAase H (but free of RNAase III). Initiation of DNA synthesis in the absence of RNAase H did not occur at the normal replication origin and was non-selective with respect to the plasmid template. In the presence of RNAase H the system was selective for ColE1-type plasmids and could not accept the DNA of non-amplifiable plasmids. Electron microscopic analysis of the reaction product formed under discriminatory conditions indicated that origin usage and directionally of ColE1, RSF1030, and CloDF13 replication were consistent with the normal replication pattern of these plasmids. It is proposed that the initiation of ColE1-type replication depends on the formation of an extensive secondary structure in the origin primer RNA that prevents its degradation by RNAase H.     

Multicomponent origin of cytomegalovirus lytic-phase DNA replication.

Cytomegalovirus (CMV) lytic-phase DNA replication requires both trans-acting factors, such as the virus-coded DNA polymerase, and a previously undefined cis-acting element, the origin, within which initiation occurs. We have located a candidate origin of CMV lytic-phase DNA replication, oriLyt, in both simian and human strains by assessing the ability of cloned restriction fragments to mediate phosphonoformic acid-sensitive DNA replication after transfection into human fibroblasts when required trans-acting factors were supplied by infection. In initial experiments the simian CMV-like strain Colburn EcoRI D fragment directed DNA replication; this fragment contains all of the single-stranded DNA-binding protein gene (dbp) and about 7 kbp of upstream sequence. A larger region upstream of human CMV dbp also mediated replication in transient assays. Subsequent subcloning and deletion analyses defined a CMV strain Colburn region sufficient for origin function, spanning about 1,300 bp in the apparently noncoding region upstream of dbp. The nucleotide sequence of this region revealed four distinct domains, containing (i) a 9-bp repeated sequence, (ii) an A+T-rich segment, (iii) an 11-bp direct repeat, and (iv) a 47-bp direct repeat. At least some part of each of these domains was required for origin function. Therefore, like the Epstein-Barr virus lytic-phase origin of DNA replication, CMV oriLyt appears to be structurally complex.     

Factor-independent activation of Escherichia coli rRNA transcription. II. characterization of complexes of rrnB P1 promoters containing or lacking the upstream activator region with Escherichia coli RNA polymerase.

A region upstream from the Escherichia coli rrnB P1 promoter, the upstream activator region (UAR), increases the activity of the promoter in vivo and the rate of association with RNA polymerase (E sigma 70) in vitro in the presence of the two initiating nucleotides. We have used four types of chemical and enzymatic footprinting probes to determine whether rrnB P1-E sigma 70 complexes formed in the presence of the initiating nucleotides (RPinit) differ from typical open complexes (RPo) formed in the absence of the initiating nucleotides and to examine the structural differences between rrnB P1 complexes containing the UAR and those lacking the UAR. We find that the rrnB P1-RPinit complex closely resembles open complexes formed at other E sigma 70 promoters, indicating that the formation of the first phosphodiester bond does not result in a major rearrangement of the promoter-RNA polymerase complex. An unusual potassium permanganate modification at position -18 in both RPo and RPinit indicates the possible presence of a subtle difference in the -10, -35 spacer structure compared to some other E. coli promoters. We show that the E sigma 70-rrnB P1 complex formed with the promoter containing the UAR has DNase I and hydroxyl radical cleavage patterns in the -50 region different from those observed with the same promoter lacking the UAR. These results are interpreted to indicate that E sigma 70 may interact with a region further upstream from that contacted by RNA polymerase bound at most other promoters and/or that unusual structural properties of this region are induced by bound E sigma 70.