Origin of adenovirus DNA replication. Role of the nuclear factor I binding site in vivo

An assay is described that detects in vivo a single round of initiation and DNA synthesis directed by a linear molecule containing an exposed single copy of an adenovirus (Ad) origin of replication. This and a previously described assay, which measures multiple rounds of DNA replication, were used to identify DNA sequences within the Ad2 and Ad4 origins of replication that are important for ori function. Linear DNA molecules containing sequences from the Ad2 or Ad4 genome termini were cotransfected with homologous and heterologous helper virus, and net amounts of DNA synthesis were compared. Linear molecules containing the Ad4 inverted terminal repeats were replicated 20-fold better in the presence of the homologous helper, whereas both Ad2 and Ad4 inverted terminal repeats were utilized efficiently by Ad4. DNA sequence analysis of the Ad2 ori and the corresponding region in Ad4 indicated that, although there are only ten variant base-pairs, eight are located within the Ad2 DNA sequence recognized by the cellular protein nuclear factor I. This protein is required to achieve the maximal rate of Ad2 DNA replication in vitro, and these differences therefore identify DNA sequences that are crucial to Ad2 ori function. The Ad4 ITR does not contain a functional nuclear factor I binding site, and deletion analysis has demonstrated that this region of the Ad4 genome is not required for ori function. In contrast to Ad2, the DNA sequences required for the initiation of Ad4 DNA replication were shown to reside entirely within the terminal 18 base-pairs of the Ad4 inverted terminal repeat.     

Bacteriophage P4 DNA replication. Nucleotide sequence of the P4 replication gene and the cis replication region

A 3100 base piece of DNA from the 11,500 base genome of bacteriophage P4 was analyzed for its nucleotide sequence. This segment of DNA contains two open reading frames of 106 and 777 amino acid residues; the latter of which is the coding sequence for the Mr 84,841 alpha protein, which is necessary for P4 DNA replication and is thought to act as a P4-specific DNA primase. A region of about 300 base-pairs localized just beyond the alpha gene and about 4500 bases from the origin of replication (ori), was defined as the locus for P4's cis replication region (crr). This region is required for replication both in vivo and in vitro, and consists of two directly repeated sequences of 120 base-pairs that match one another at 98 positions. These directly repeated sequences are separated by 60 base-pairs, which are not necessary for replication. Each repeat in crr contains three copies of the octamer TGTTCACC that is found six times in ori. Either of the 120 base-pair repeat sequences in crr is sufficient for replication, and the entire crr can function in an inverted orientation. crr is also active at a distance of 1800 bases from the P4 origin of replication.     

Replication-specific conversion of the Staphylococcus aureus pT181 initiator protein from an active homodimer to an inactive heterodimer.

The Staphylococcus aureus rolling circle plasmid pT181 regulates its replication by controlling the synthesis of its initiator protein RepC. RepC is inactivated during pT181 replication by the addition of an oligodeoxynucleotide, giving rise to a new form, RepC*. We analyzed RepC and RepC* in four classes of mutants: plasmid copy number mutants, two classes of RepC mutants affecting different portions of the protein and oriC (origin) mutants. We have found that in the cell with wild-type RepC there are similar relative amounts of RepC and RepC*, regardless of copy number, and that the conversion of RepC to RepC* is replication dependent. Genetic and biochemical evidence is presented that RepC functions as a dimer and that during replication the RepC homodimer is converted to the RepC/RepC* heterodimer.     

Specificity of RepC protein in plasmid pT181 DNA replication

The plasmid pT181 of Staphylococcus aureus consists of 4437 base pairs and encodes resistance to tetracycline. Initiation of pT181 DNA replication specifically requires the plasmid-encoded initiator protein, RepC. The initiator protein binds specifically to a 32-base pair sequence within the pT181 origin of replication. RepC protein also has a nicking-closing activity that is specific for the pT181 origin. Replication of pT181 initiates by covalent extension of the nick and proceeds by a rolling circle mechanism. Two other small, multicopy plasmids pC221 and pS194 belong to the pT181 family and have common structural organization and replication properties. The replication proteins and replication origins of these plasmids have extensive sequence homologies, although they belong to different incompatibility groups. In spite of this homology, the replication proteins and replication origins of these three plasmids do not show any cross-reactivity in vivo. We have carried out a series of in vitro experiments to determine the specificity of pT181-encoded initiator protein, RepC. DNA binding experiments showed that although the binding of RepC to the pT181 origin was very efficient, little or no binding was seen with pC221 and pS194 origins. The nicking-closing activity of RepC was found to be equally efficient with the pC221 and pS194 plasmids. The plasmids pC221 and pS194 replicated efficiently in a RepC-dependent in vitro system. However, replication of these plasmids was greatly reduced in the presence of a competing pT181 origin. The results presented here suggest that nicking-closing by RepC at the origin is not sufficient for maximal replication and that tight binding of RepC to the origin plays an important role in the initiation of DNA replication.     

Mutational analysis of the bacteriophage phi X174 replication origin

Bacteriophage phi X174 mutants within the 30 base-pair replication origin were constructed using oligodeoxynucleotide-directed mutagenesis. A total of 18 viable base substitution mutants at 13 different positions within the origin region were obtained. The majority of these ori mutants have a plaque morphology and burst size comparable to that of wild-type phi X174. Two phi X174 ori mutants with a reduced growth ability spontaneously acquired additional mutations that enhanced the growth rate. The additional mutation was located at the same site as the original mutation or was located in the N-terminal part of the gene A protein. This latter secondary mutation is responsible for a better binding and/or recognition of the gene A protein to the mutated origin. In a Darwinian experiment wild-type phi X174 outgrows all phi X174 ori mutants, indicating the superiority of the wild-type ori sequence for the reproduction of bacteriophage phi 174. Insertions and deletions were constructed at different positions within the phi X174 replication origin cloned in a plasmid. Small insertions and deletions in the A + T-rich spacer region do not inhibit phi X174 gene A protein cleavage in vitro, but severely impair packaging of single-stranded plasmid DNA in viral coats.     

Structure of replication origin of the Escherichia coli K-12 chromosome: the presence of spacer sequences in the ori region carrying information for autonomous replication.

The replication origin region of the Escherichia coli K-12 chromosome has been cloned, and a region of 245 base-pairs has been shown to contain all the information for autonomous replication (defined ori). In order to obtain further information on the sequence organization in the defined ori stretch, various types of mutation were introduced by in vitro techniques at a restriction site (AvaII site) which locates near the middle of ori. When the correlation between these mutations and replicating function was examined, different effects were obtained with the types of mutation: the replicating function was completely destroyed by either insertion or deletion of short sequences, but not by base-substitutions. Based on these observations and on the fact that multi-gene products are involved in the initiation of replication, we assume that two categories of sequences are present within the ori stretch, one specifying interaction with initiation factors (recognition sequences) and the other spacing the recognition sequences in appropriate distances (spacer sequences), and that the AvaII site is located in the spacer region.     

The interaction of RepC initiator with iterons in the replication of the broad host-range plasmid RSF1010.

The replication origin of the broad host-range plasmid RSF1010 contains 3.5 copies of a 20mer iteron sequence that bind specifically to the plasmid-encoded initiator, RepC. Here we demonstrated that even a single iteron was bent upon binding of RepC. Moreover, the bending angle seems to become larger along with the increment of the number of iterons. In a mutational analysis of the iteron sequence, we isolated seven kinds of base-substitution mutants of iterons, and estimated the replication activity of these mutants in vivo. We found that each of the subsections in the 20mer iteron sequence made a distinct contribution to the initiation of RSF1010 DNA replication. With the binding assay of RepC and mutated iterons in vitro, we found that the formation of a productive RepC-iteron complex was required for the initiation of plasmid DNA replication.     

Characterization and comparative sequence analysis of replication origins from three large Bacillus thuringiensis plasmids.

The replication origins of three large Bacillus thuringiensis plasmids, derived from B. thuringiensis HD263 subsp. kurstaki, have been cloned in Escherichia coli and sequenced. The replication origins, designated ori 43, ori 44, and ori 60, were isolated from plasmids of 43, 44, and 60 MDa, respectively. Each cloned replication origin exhibits incompatibility with the resident B. thuringiensis plasmid from which it was derived. Recombinant plasmids containing the three replication origins varied in their ability to transform strains of B. thuringiensis, Bacillus megaterium, and Bacillus subtilis. Analysis of the derived nucleotide and amino acid sequences indicates that the replication origins are nonhomologous, implying independent derivations. No significant homology was found to published sequences of replication origins derived from the single-stranded DNA plasmids of gram-positive bacteria, and shuttle vectors containing the three replication origins do not appear to generate single-stranded DNA intermediates in B. thuringiensis. The replication origin regions of the large plasmids are each characterized by a single open reading frame whose product is essential for replication in B. thuringiensis. The putative replication protein of ori 60 exhibits partial homology to the RepA protein of the Bacillus stearothermophilus plasmid pTB19. The putative replication protein of ori 43 exhibits weak but extensive homology to the replication proteins of several streptococcal plasmids, including the open reading frame E replication protein of the conjugative plasmid pAM beta 1. The nucleotide sequence of ori 44 and the amino acid sequence of its putative replication protein appear to be nonhomologous to other published replication origin sequences.     

Control of pT181 replication I. The pT181 copy control function acts by inhibiting the synthesis of a replication protein

pT181 is a fully sequenced 4.4-kb 20 copy Tcr plasmid from Staphylococcus aureus. Its replication system involves a unique unidirectional origin embedded in the coding sequence for a plasmid-determined protein, RepC, that is required for initiation. When joined to a 55 copy carrier plasmid, pE194, pT181 excludes autonomous isologous replicons by inhibiting their replication. Two types of spontaneous pT181 copy mutants have been isolated, one that eliminates sensitivity to this inhibition and another that does not. A spontaneous 180-bp deletion, delta 144, eliminates both the inhibitory activity and sensitivity to it. This deletion increases copy number by 50-fold and RepC production by at least 10-fold. It is located directly upstream from the repC coding sequence and the deletion-bearing plasmid supports the replication of inhibitor-sensitive plasmids in cells containing active inhibitor. This effect is probably due to the overproduction of RepC by the delta 144 plasmid. On the basis of these results, it is suggested that RepC synthesis is negatively controlled by an inhibitor that is encoded directly upstream from the repC coding sequence and acts as a tareget set in the same region. It is likely, therefore, that pT181 replication rate is determined by the level of RepC.     

An 18-base-pair sequence is sufficient for termination of rolling-circle replication of plasmid pT181.

pT181 and related plasmids of gram-positive bacteria replicate by a rolling-circle mechanism. The replication initiator protein of pT181, RepC, has origin-specific nicking-closing activities. Replication of the plasmid pT181 leading strand initiates by covalent extension of the RepC-generated nick, and the origin of replication contains signals for both initiation and termination of DNA replication. We have investigated the sequence requirements for the initiation and termination steps by using plasmids containing two pT181 origins. In vitro replication experiments showed that 18- and 24-bp synthetic oligonucleotides containing the RepC nick site were active in the termination of replication. However, initiation of replication required a larger region which also includes the RepC binding site. Plasmids containing the 18- and 24-bp region were also found to be nicked by the RepC protein. Our results demonstrate that sequence requirements for initiation and termination of pT181 replication overlap, but while the RepC binding site is required for initiation, it is dispensable for termination.     

Cleavage of single-stranded DNA by plasmid pT181-encoded RepC protein.

RepC protein encoded by plasmid pT181 has single-stranded endonuclease and topoisomerase-like activities. These activities may be involved in the initiation (and termination) of pT181 replication by a rolling circle mechanism. RepC protein cleaves the bottom strand of DNA within the origin of replication at a single, specific site when the DNA is in the supercoiled or linear (double or single-stranded) form. We have found that RepC protein will also cleave single-stranded DNA at sites other than the origin of replication. We have mapped the secondary cleavage sites on pT181 DNA. When the DNA is in the supercoiled, or linear, double-stranded form, only the primary site within the origin is cleaved. However, when the DNA is present in the single-stranded form, several strong and weak cleavage sites are observed. The DNA sequence at these cleavage sites shows a strong similarity with the primary cleavage site. The presence of Escherichia coli SSB protein inhibited cleavage at all of the secondary nick sites while the primary nick site remained susceptible to cleavage.     

In vitro studies of the initiation of staphylococcal plasmid replication. Specificity of RepD for its origin (oriD) and characterization of the Rep-ori tyrosyl ester intermediate

Several staphylococcal plasmids from different incompatibility (inc) groups which replicate by a rolling circle mechanism each specify a replication initiator protein (Rep) which is homologous with that of the inc3 tetracycline resistance plasmid pT181. The rep gene sequences of six pT181-like plasmids are known, each encoding proteins of molecular mass 38 kDa with 62% overall amino acid sequence identity. The initiation of replication in vivo by each of the Rep proteins is plasmid specific, acting in trans only at the cognate replication origin (ori) of the encoding plasmid. Previous studies in vitro of the RepC protein of pT181 demonstrated replication initiator, topoisomerase-like, and DNA binding activities, which appeared to be specific for the origin (oriC) of pT181 when compared with unrelated staphylococcal plasmids. Although RepD, specified by the inc4 chloramphenicol resistance plasmid pC221, has a range of activities similar to those noted previously for RepC, manipulation of in vitro conditions has revealed discrete steps in the overall reaction of RepD with oriD. In addition, factors have been identified which are necessary not only for sequence-dependent discrimination in vitro by Rep proteins for all pT181-like plasmids but also for the absolute specificity of RepD for its cognate pC221 replication origin (oriD), the latter occurring in vivo and a function of the topological state of the ori-containing target DNA. Here we also demonstrate the presence of a covalent phosphoryl-tyrosine linkage between the RepD protein of plasmid pC221 and an oligonucleotide substrate corresponding to its replication origin (oriD). The reactive tyrosine (Tyr-188) was identified from amino acid sequences of 32P-labeled peptide-oligonucleotide fragments. Substitution of Tyr-188 with phenylalanine confirms the importance of the tyrosyl hydroxyl group since the Y188F protein retains the sequence-specific DNA-binding capabilities of wild-type RepD but is unable to attach covalently to the replication origin or participate in the nicking-closing reaction in vitro.     

Plasmid R6K DNA replication. II. Direct nucleotide sequence repeats are required for an active gamma-origin

Insertions of the transposable element Tn5 were obtained at a number of sites within the γ origin region of plasmid R6K. A contiguous region of DNA, 240 base-pairs in length, was identified as the functional γ origin of replication on the basis that Tn5 insertions within this region inactivated the origin. This region contains seven, tandemly arranged, 22 base-pair direct repeats. Spontaneous deletion of a Tn5 sequence that is inserted in one of the repeats was accompanied generally by the loss of nucleotide sequences equivalent to one or more of the repeat sequences. Deletion mutants of the γ origin that lack one or two repeats are still functional but a plasmid containing such a mutant origin is not maintained stably. Deletions of nucleotide sequences equivalent to four or more of the direct repeats result in the inactivation of the R6K γ origin.     

Pivotal role of the non-hr origin of DNA replication in the genesis of defective interfering baculoviruses

The generation of deletion mutants, including defective interfering viruses, upon serial passage of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) in insect cell culture has been studied. Sequences containing the non-homologous region origin of DNA replication (non-hr ori) became hypermolar in intracellular viral DNA within 10 passages in Se301 insect cells, concurrent with a dramatic drop in budded virus and polyhedron production. These predominant non-hr ori-containing sequences accumulated in larger concatenated forms and were generated de novo as demonstrated by their appearance and accumulation upon infection with a genetically homogeneous bacterial clone of SeMNPV (bacmid). Sequences were identified at the junctions of the non-hr ori units within the concatemers, which may be potentially involved in recombination events. Deletion of the SeMNPV non-hr ori using RecE/RecT-mediated homologous ET recombination in Escherichia coli resulted in a recombinant bacmid with strongly enhanced stability of virus and polyhedron production upon serial passage in insect cells. This suggests that the accumulation of non-hr oris upon passage is due to the replication advantage of these sequences. The non-hr ori deletion mutant SeMNPV bacmid can be exploited as a stable eukaryotic heterologous protein expression vector in insect cells.     

Interaction between the replication origin and the initiator protein of the filamentous phage f1. Binding occurs in two steps

The replication initiator protein of bacteriophage f1 (gene II protein) binds to the phage origin and forms two complexes that are separable by polyacrylamide gel electrophoresis. Complex I is formed at low gene II protein concentrations, and shows protection from DNase I of about 25 base-pairs (from position +2 to +28 relative to the nicking site) at the center of the minimal origin sequence. Complex II is produced at higher concentrations of the protein, and has about 40 base-pairs (from -7 to +33) protected. On the basis of gel mobility, complex II appears to contain twice the amount of gene II protein as does complex I. The 40 base-pair sequence protected in complex II corresponds to the minimal origin sequence as determined by in-vivo analyses. The central 15 base-pair sequence (from +6 to +20) of the minimal origin consists of two repeats in inverted orientation. This sequence, when cloned into a plasmid, can form complex I, but not complex II. We call this 15 base-pair element the core binding sequence for gene II protein. Methylation interference with the formation of complex I by the wild-type origin indicates that gene II protein contacts six guanine residues located in a symmetric configuration within the core binding sequence. Formation of complex II requires, in addition to the core binding sequence, the adjacent ten base-pair sequence on the right containing a third homologous repeat. A methylation interference experiment performed on complex II indicates that gene II protein interacts homologously with the three repeats. In complex II, gene II protein protects from DNase I digestion not only ten base-pairs on the right but also ten base-pairs on the left of the sequence that is protected in complex I. Footprint analyses of various deletion mutants indicate that the left-most ten base-pairs are protected regardless of their sequence. The site of nicking by gene II protein is located within this region. A model is presented for the binding reaction involving both protein-DNA and protein-protein interactions.     

DNA replication and chromatin structure of simian virus 40 insertion mutants.

Insertion of DNA segments into the nuclease-sensitive region of simian virus 40 alters both replication efficiency and chromatin structure. Mutants containing large insertions between the simian virus 40 origin of replication (ori site) and the 21-base-pair repeated sequences replicated poorly when assayed by transfection into COS-1 cells. Replication of mutants with shorter insertions was moderately reduced. This effect was cis-acting and independent of the nucleotide sequence of the insert. The nuclease-sensitive chromatin structure was retained in these mutants, but the pattern of cleavage sites was displaced in the late direction from the ori site. New cleavage sites appeared within the inserted sequences, suggesting that information specifying the nuclease-sensitive chromatin structure is located on the late side of the inserts. Accessibility to BglI (which cleaves within the ori site) was reduced in the larger insertion mutants. These results support the conclusion that efficient function of the viral origin of replication is correlated with its proximity to an altered chromatin structure.     

An enhancer of DNA replication.

cmp, a nucleotide sequence element in the plasmid pT181 of Staphylococcus aureus, acts as an enhancer of DNA replication. When cmp is present on an unrelated vector along with the pT181 origin of replication, it increases the ability of the linked pT181 origin to compete with a coresident pT181 plasmid for the initiator protein RepC. cmp is contained within a 156-base-pair segment, and its deletion from pT181 reduces by twofold the frequency of plasmid replication under derepressed conditions. The enhancer sequence contains a locus of DNA bending, and enhancer activity decreases with distance from the replication origin.