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.     

Unidirectional replication of plasmid R100

We isolated a 284 base-pair BamHI fragment of plasmid R100 that supports initiation of replication of a plasmid regardless of the orientation of the fragment. Analysis of the specific radioactivity of restriction fragments from 32P-labeled replication intermediates synthesized in vitro shows that replication of the plasmid carrying the 284 base-pair fragment is unidirectional. The direction of replication depends on the orientation of the fragment present in the plasmid. The 5' ends of the leading-strand DNA formed in the early stage of replication were mapped to a region downstream from the 284 base-pair fragment in the direction of replication. The lagging-strand DNA products were also identified and their 3' ends mapped to unique sites within the 284 base-pair fragment causing unidirectional replication of R100.     

Replication initiator protein mRNA of ColE2 plasmid and its antisense regulator RNA are under the control of different degradation pathways

Replication of the ColE2 plasmid requires a plasmid-coded initiator protein (Rep). Rep expression is controlled by antisense RNA (RNAI) against the Rep mRNA at a translational step. In this paper, we examined the effects of host RNA degradation enzymes on the degradation process of the Rep mRNA and its degradation intermediates especially those carrying the 5' untranslated region. We showed that the Rep mRNA is subjected to complex degradation pathways involving at least RNase I, RNase II, RNase III, RNase E, RNase G and PNPase. RNase II acts as a major exoribonuclease and PNPase plays a minor role. We also showed that the PcnB (polyA polymerase I) plays only a minor role in the Rep mRNA degradation process. The RNA degradation pathways of the Rep mRNA and RNAI of the ColE2 plasmid are quite different. Based on these results, we speculate that the ColE2 Rep mRNA and RNAI are endowed with individual RNA half lives required for the efficient copy number control by being subjected to different RNA degradation systems.     

The Rep protein binding elements of the plasmid ColE2-P9 replication origin

The plasmid ColE2-P9 (ColE2) origin (32bp) is specifically recognized by the plasmid-specified Rep protein that initiates DNA replication. The ColE2 origin is divided into at least three functional subregions (I, II, and III), and three sites (a, b, and c) found in subregions I and II play important roles in Rep protein binding. We performed SELEX experiments of plasmid ColE2 to determine the optimal sequences for specific binding of the Rep protein. From these experiments, we obtained a common 16-bp sequence (5'-TGAGACCANATAAGCC-3'), which corresponds to about one half of the minimal ColE2 origin and contains sites a and b. Gel mobility shift assays using single-point mutant origins and the Rep protein further indicated that high affinity sequence-specific recognition by the Rep protein requires sites a, b, and c, but that mutations in site c were less disruptive to this recognition than those in sites a and b.     

Replication of ColE2 and ColE3 plasmids In vitro replication dependent on plasmid-coded proteins

Summary We developed an in vitro replication system for ColE2 and ColE3 plasmids using cell extracts prepared from bacteria with or without these plasmids. DNA synthesis depended on host DNA polymerase I and was sensitive to rifampicin and chloramphenicol. Preincubation of the extracts with plasmid DNA, however, allowed replication of template DNA added subsequently in a plasmid-specific manner in the presence of rifampicin and chloramphenicol. The plasmid-specified trans-acting factor(s) was detected in cell extracts from bacteria carrying a recombinant plasmid with the region of ColE2 or ColE3 encoding the Rep protein. The plasmid-specified factor(s) consisted at least in part of protein, probably the Rep protein. In vitro replication started within a region of ColE2 or ColE3 containing the smallest cis-acting segment essential for in vivo replication and proceeded in a fixed direction.     

The effects of RNA degradation enzymes on antisense RNAI controlling ColE2 plasmid copy number

Replication of the ColE2 plasmid requires a plasmid-coded initiator protein (Rep). Rep expression is controlled by antisense RNA (RNAI), which prevents the Rep mRNA translation. In this paper, we examined the effects of RNA degradation enzymes on the degradation pathways of RNAI of the ColE2 plasmid. In the DeltapcnB strain lacking the poly(A) polymerase I (PAP I) the RNAI degradation intermediate (RNAI(*)) accumulates much more than that in the wt strain. RNAI(*) is produced by the RNase E cleavage. RNase II and PNPase are involved in further degradation of RNAI(*) and PAP I is necessary for efficient degradation. The degradation process of ColE2 RNAI is similar to those of R1 CopA RNA and ColE1 RNAI, although the nucleotide sequences and fine secondary structures of these three RNAs are different. ColE2 RNAI is cleaved at multiple positions in the 5' end region by RNase E. The degradation pathway of ColE2 RNAI shown here is quite different from that of the ColE2 Rep mRNA which we have previously reported. In the DeltapcnB strain used for RNA analysis the copy number of the ColE2 plasmid decreases to about a half as compared with that in the isogenic wt strain.     

Anatomy of the replication origin of plasmid ColE2-P9

The plasmid ColE2-P9 origin is a 32-bp region which is specifically recognized by the plasmid-specified Rep protein to initiate DNA replication. We analyzed the structural and functional organization of the ColE2 origin by using various derivatives carrying deletions and single-base-pair substitutions. The origin may be divided into three subregions: subregion I, which is important for stable binding of the Rep protein; subregion II, which is important for binding of the Rep protein and for initiation of DNA replication; and subregion III, which is important for DNA replication but apparently not for binding of the Rep protein. The Rep protein might recognize three specific DNA elements in subregions I and II. The relative transformation frequency of the autonomously replicating plasmids carrying deletions in subregion I is lower, and nevertheless the copy numbers of these plasmids in host bacteria are higher than those of the wild-type plasmid. Efficient and stable binding of the Rep protein to the origin might be important for the replication efficiency to be at the normal (low) level. Subregion II might be essential for interaction with the catalytic domain of the Rep protein for primer RNA synthesis. The 8-bp sequence across the border of subregions II and III, including the primer sequence, is conserved in the (putative) origins of many plasmids, the putative Rep proteins of which are related to the ColE2-P9 Rep protein. Subregion III might be required for a step that is necessary after Rep protein binding has taken place.     

Mapping initiation sites for simian virus 40 DNA synthesis events in vitro.

Primer RNA-DNA, a small (approximately 30-nucleotide) RNA-DNA hybrid molecule, was identified in recent studies of simian virus 40 DNA synthesis in vitro. The available evidence indicates that primer RNA-DNA is the product of the polymerase alpha-primase complex. Primer RNA-DNA is formed exclusively on lagging-strand DNA templates; it is synthesized initially in the vicinity of the simian virus 40 origin and at later times at sites progressively distal to the origin. To further characterize initiation events, template sequences encoding the 5' ends of both primer RNA and primer DNA, formed during a 5-s pulse, have been determined. Analyses of these sequences demonstrate the existence of an initiation signal for lagging-strand synthesis. At any given position, the initiation signal is located within those template sequences encoding primer RNA, situated proximal to the nucleotide encoding the 5' end of the RNA primer. In most instances, the sequence 5'-TTN-3' (where N encodes the nucleotide at the 5' end of the primer) is a feature of the initiation signal. Initiation signals are present, on average, once every 19 nucleotides. These results are discussed in terms of the mechanism of Okazaki fragment formation and possible links between prokaryotic and eukaryotic initiation events.     

Rolling-circle replication of bacterial plasmids.

Many bacterial plasmids replicate by a rolling-circle (RC) mechanism. Their replication properties have many similarities to as well as significant differences from those of single-stranded DNA (ssDNA) coliphages, which also replicate by an RC mechanism. Studies on a large number of RC plasmids have revealed that they fall into several families based on homology in their initiator proteins and leading-strand origins. The leading-strand origins contain distinct sequences that are required for binding and nicking by the Rep proteins. Leading-strand origins also contain domains that are required for the initiation and termination of replication. RC plasmids generate ssDNA intermediates during replication, since their lagging-strand synthesis does not usually initiate until the leading strand has been almost fully synthesized. The leading- and lagging-strand origins are distinct, and the displaced leading-strand DNA is converted to the double-stranded form by using solely the host proteins. The Rep proteins encoded by RC plasmids contain specific domains that are involved in their origin binding and nicking activities. The replication and copy number of RC plasmids, in general, are regulated at the level of synthesis of their Rep proteins, which are usually rate limiting for replication. Some RC Rep proteins are known to be inactivated after supporting one round of replication. A number of in vitro replication systems have been developed for RC plasmids and have provided insight into the mechanism of plasmid RC replication.     

Interaction between the T4 helicase-loading protein (gp59) and the DNA polymerase (gp43): a locking mechanism to delay replication during replisome assembly

The T4 helicase-loading protein (gp59) has been proposed to coordinate leading- and lagging-strand DNA synthesis by blocking leading-strand synthesis during the primosome assembly. In this work, we unambiguously demonstrate through a series of biochemical and biophysical experiments, including single-molecule fluorescence microscopy, that the inhibition of leading-strand holoenzyme progression by gp59 is the result of a complex formed between gp59 and leading-strand polymerase (gp43) on DNA that is instrumental in preventing premature replication during the assembly of the T4 replisome. We find that both the polymerization and 3' --> 5' exonuclease activities of gp43 are totally inhibited within this complex. Chemical cross-linking of the complex followed by tryptic digestion and peptide identification through matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry identified Cys169 of gp43 and Cys215 of gp59 as residues in a region of a protein-protein contact. With the available crystal structures for both gp43 and gp59, a model of the complex was constructed based on shape complementarity, revealing that parts of the C-terminal domain from gp59 insert into the interface created by the thumb and exonuclease domains of gp43. This insertion effectively locks the polymerase into a conformation where switching between the pol and editing modes is prevented. Thus, continued assembly of the replisome through addition of the primosome components and elements of the lagging-strand holoenzyme can occur without leading-strand DNA replication.     

Functional uncoupling of twin polymerases: mechanism of polymerase dissociation from a lagging-strand block

Replication forks are constantly subjected to events that lead to fork stalling, stopping, or collapse. Using a synthetic rolling circle DNA substrate, we demonstrate that a block to the lagging-strand polymerase does not compromise helicase or leading-strand polymerase activity. In fact, lagging-strand synthesis also continues. Thus, the blocked lagging-strand enzyme quickly dissociates from the block site and resumes synthesis on new primed sites. Furthermore, studies in which the lagging polymerase is continuously blocked show that the leading polymerase continues unabated even as it remains attached to the lagging-strand enzyme. Hence, upon encounter of a block to the lagging stand, the polymerases functionally uncouple yet remain physically associated. Further study reveals that naked single-stranded DNA results in disruption of a stalled polymerase from its beta-DNA substrate. Thus, as the replisome advances, the single-stranded DNA loop that accumulates on the lagging-strand template releases the stalled lagging-strand polymerase from beta after SSB protein is depleted. The lagging-strand polymerase is then free to continue Okazaki fragment production.     

Timing, Coordination, and Rhythm: Acrobatics at the DNA Replication Fork

In DNA replication, the antiparallel nature of the parental duplex imposes certain constraints on the activity of the DNA polymerases that synthesize new DNA. The leading-strand polymerase advances in a continuous fashion, but the lagging-strand polymerase is forced to restart at short intervals. In several prokaryotic systems studied so far, this problem is solved by the formation of a loop in the lagging strand of the replication fork to reorient the lagging-strand DNA polymerase so that it advances in parallel with the leading-strand polymerase. The replication loop grows and shrinks during each cycle of Okazaki fragment synthesis. The timing of Okazaki fragment synthesis and loop formation is determined by a subtle interplay of enzymatic activities at the fork. Recent developments in single-molecule techniques have enabled the direct observation of these processes and have greatly contributed to a better understanding of the dynamic nature of the replication fork. Here, we will review recent experimental advances, present the current models, and discuss some of the exciting developments in the field.     

Comparative analysis of the replicon regions of eleven ColE2-related plasmids.

The incA gene product of ColE2-P9 and ColE3-CA38 plasmids is an antisense RNA that regulates the production of the plasmid-coded Rep protein essential for replication. The Rep protein specifically binds to the origin and synthesizes a unique primer RNA at the origin. The IncB incompatibility is due to competition for the Rep protein among the origins of the same binding specificity. We localized the regions sufficient for autonomous replication of 15 ColE plasmids related to ColE2-P9 and ColE3-CA38 (ColE2-related plasmids), analyzed their incompatibility properties, and determined the nucleotide sequences of the replicon regions of 9 representative plasmids. The results suggest that all of these plasmids share common mechanisms for initiation of DNA replication and its control. Five IncA specificity types, 4 IncB specificity types, and 9 of the 20 possible combinations of the IncA and IncB types were found. The specificity of interaction of the Rep proteins and the origins might be determined by insertion or deletion of single nucleotides and substitution of several nucleotides at specific sites in the origins and by apparently corresponding insertion or deletion and substitution of amino acid sequences at specific regions in the C-terminal portions of the Rep proteins. For plasmids of four IncA specificity types, the nine-nucleotide sequences at the loop regions of the stem-loop structures of antisense RNAs are identical, suggesting an evolutionary significance of the sequence. The mosaic structures of the replicon regions with homologous and nonhomologous segments suggest that some of them were generated by exchanging functional parts through homologous recombination.     

Distinct functions of the two specificity determinants in replication initiation of plasmids ColE2-P9 and ColE3-CA38

The plasmid ColE2-P9 Rep protein specifically binds to the cognate replication origin to initiate DNA replication. The replicons of the plasmids ColE2-P9 and ColE3-CA38 are closely related, although the actions of the Rep proteins on the origins are specific to the plasmids. The previous chimera analysis identified two regions, regions A and B, in the Rep proteins and two sites, alpha and beta, in the origins as specificity determinants and showed that when each component of the region A-site alpha pair and the region B-site beta pair is derived from the same plasmid, plasmid DNA replication is efficient. It is also indicated that the replication specificity is mainly determined by region A and site alpha. By using an electrophoretic mobility shift assay, we demonstrated that region B and site beta play a critical role for stable Rep protein-origin binding and, furthermore, that 284-Thr in this region of the ColE2 Rep protein and the corresponding 293-Trp of the ColE3 Rep protein mainly determine the Rep-origin binding specificity. On the other hand, region A and site alpha were involved in the efficient unwinding of several nucleotide residues around site alpha, although they were not involved in the stable binding of the Rep protein to the origin. Finally, we discussed how the action of the Rep protein on the origin involving these specificity determinants leads to the plasmid-specific replication initiation.     

Novel RNA family structure of hepatitis B virus expressed in human cells, using a helper-free adenovirus vector.

Ad5-HBL is a type 5 adenovirus bearing the large BglII fragment (2.8 kilobases; 87% of the total genome) of hepatitis B virus (HBV), subtype adr. Eight HBV RNAs expressed in HeLa cells infected with Ad5-HBL were mapped by the nuclease S1 technique. Three major RNAs spanning 2.4, 2.0, and 0.7 kilobases of the HBV sequences cover the coding regions of "presurface" plus surface antigen, surface antigen alone, and "X" protein, respectively. The 5' segment of an RNA which could code for core antigen (HBcAg) was also detected. All major HBV RNAs initiate from mutually exclusive 5' ends, terminate at the unique 3' end within the HBcAg coding region (except readthrough species), and have no spliced deletion, forming a novel RNA family structure. No TATA box-like sequences were found near the 5' end of these RNAs, except in the case of the 2.4-kilobase RNA. About two thirds of total HBV RNA does not terminate at the mapped 3'-end position, suggesting the termination signal is functionally inefficient. Since the potential 5' end of HBcAg mRNA was mapped at the same position as the minus-strand nick of HBV DNA previously reported, we propose a model that requires inefficient poly(A) addition to produce an RNA which serves both as HBcAg mRNA and as the putative RNA template of minus-strand DNA synthesis in the HBV life cycle.     

Isolation and characterization of pHW15, a small cryptic plasmid from Rahnella genomospecies 2

A small cryptic plasmid designated pHW15 was isolated from Rahnella genomospecies 2 WMR15 and its complete nucleotide sequence was determined. The plasmid contained 3002 bp with a G+C content of 47.4%. The origin of replication was identified by deletion analysis as a region of about 600 bp. This region had an identity of 70% to the replication origin of the ColE1 plasmid at the nucleotide level. Sequence analysis revealed the typical elements: RNA I, RNA II and their corresponding promoters, a sequence allowing hybridisation of RNA II to the DNA and favouring processing by RNaseH, a single-strand initiation determinant (ssi) that allows initiation of lagging-strand synthesis, and a terH sequence required for termination of lagging-strand synthesis. The plasmid contained three expressed open reading frames, one of which showed homology to a ColE1 plasmid-encoded protein. Furthermore, a multimer resolution site was identified by sequence analysis. Its deletion resulted in formation of plasmid multimers during growth leading to an increased plasmid loss rate.     

Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork. IV. Reconstitution of an asymmetric, dimeric DNA polymerase III holoenzyme.

Individually purified subunits have been used to reconstitute the action of the Escherichia coli DNA polymerase III holoenzyme (Pol III HE) at a replication fork formed in the presence of the primosome, the single-stranded DNA binding protein, and a tailed form II DNA template. Complete activity, indistinguishable from that of the intact DNA Pol III HE, could be reproduced with a combination of the DNA polymerase III core (Pol III core), the gamma.delta complex, and the beta subunit. Experiments where the Pol III core in reaction mixtures containing active replication forks was diluted suggested that the lagging-strand Pol III core remained associated continuously with the replication fork through multiple cycles of Okazaki fragment synthesis. Since the lagging-strand Pol III core must dissociate from the 3' end of the completed Okazaki fragment, this suggests that its association with the fork is via protein-protein interactions, lending credence to the idea that it forms a dimeric complex with the leading-strand Pol III core. An asymmetry in the action of the subunits was revealed under conditions (high ionic strength) that were presumably destabilizing to the integrity of the replication fork. Under these conditions, tau acted to stimulate DNA synthesis only when the primase was present (i.e. when lagging-strand DNA synthesis was ongoing). This stimulation was reflected by an inhibition of the formation of small Okazaki fragments, suggesting that, within the context of the model developed to account for the temporal order of steps during a cycle of Okazaki fragment synthesis, the presence of tau accelerated the transit of the lagging-strand Pol III core from the 3' end of the completed Okazaki fragment to the 3' end of the new primer.     

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.