Single-strand Origin Research Articles

Three Single-Strand Origins Located on Both Strands of theStreptomycesRolling Circle Plasmid pSN22

pSN22 is an 11-kbp, high-copy-numberStreptomycesplasmid which replicates via a single-stranded intermediate by the rolling circle replication (RCR) mechanism. We identified an unidirectional single-strand origin (SSO) of pSN22,sso1,where the initiation of second-strand synthesis takes place, located between thespdAandtraRgenes in a noncoding region which is functional in its natural orientation. The nucleotide sequence ofsso1is similar over 170 bp to the SSOs of theStreptomycesplasmids pIJ101 and pJV1. A previous report described that a 548-bpBglII–SmaI fragment has an SSO activity (ori2;Kataokaet al., Mol. Gen. Genet.242,130–136, 1994). To our surprise, we discovered that on pSN22, the SSO in theBglII–SmaI fragment is in the wrong, inactive, orientation and thus cannot be involved in the conversion of the single-stranded pSN22 replication intermediate to the double-stranded form of the plasmid. We revealed that thisBglII–SmaI fragment contains two SSO fragments. Secondary structure analysis of these two SSOs showed similarity to the consensus TAGCGT which is conserved in SSOs of RCR plasmids fromStaphylococcusand the other several Gram-positive bacteria. Deletion of these hexanucleotide sequences caused loss of SSO activities. Our result shows that two types of SSOs,Streptomycestype andStaphylococcus-like type, can function inStreptomyces lividans.

Sequence of plasmid pGT5 from the archaeon Pyrococcus abyssi: evidence for rolling-circle replication in a hyperthermophile

The plasmid pGT5 (3,444 bp) from the hyperthermophilic archaeon Pyrococcus abyssi GE5 has been completely sequenced. Two major open reading frames with a good coding probability are located on the same strand and cover 85% of the total sequence. The larger open reading frame encodes a putative polypeptide which exhibits sequence similarity with Rep proteins of plasmids using the rolling-circle mechanism for replication. Upstream of this open reading frame, we have detected an 11-bp motif identical to the double-stranded origin of several bacterial plasmids that replicate via the rolling-circle mechanism. A putative single-stranded origin exhibits similarities both to bacterial primosome-dependent single-stranded initiation sites and to bacterial primase (dnaG) start sites. A single-stranded form of pGT5 corresponding to the plus strand was detected in cells of P. abyssi. These data indicate that pGT5 replicates via the rolling-circle mechanism and suggest that members of the domain Archaea contain homologs of several bacterial proteins involved in chromosomal DNA replication. Phylogenetic analysis of Rep proteins from rolling-circle replicons suggest that diverse families diverged before the separation of the domains Archaea, Bacteria, and Eucarya.

Nucleotide Sequence and Analysis of pWC1, A pC194-Type Rolling Circle Replicon inLactococcus lactis

A 2.8-kb cryptic plasmid showing no homology to either pFX3 (rolling circle, pE194-type) or pCI305 (theta-type) lactococcal replicons was identified inLactococcus lactissubsp.cremoris2204. The plasmid, pWC1, was compatible with both pCI3340 (a pCI305 derivative) and pFX3 inL. lactissubsp.cremoris2204. Sequence analysis of pWC1 showed one major ORF encoding a protein with a deduced size of 316 amino acids (aa). Database comparisons showed that the protein was distinct from the pFX- and pCI-type replication proteins (less than 21% aa identity), but shared significant homology (up to 57% aa identity) with the replication proteins from a different group of rolling circle plasmids (pC194-type) commonly found in gram-positive bacteria. A pC194-type rolling circle plasmid has not been previously described inL. lactis.Further sequence analysis showed a conserved double-stranded origin of replication in pWC1 preceded by a large (118-bp) direct repeat. The chloramphenicol-resistance gene from pC194 was inserted into a nonessential region of pWC1 to give pCP12. The host range of pCP12 includedStreptococcus thermophilus, Enterococcus faecalis,andStaphylococcus aureus,but notEscherichia coli.Both pCP12 and to a lesser extent pWC1 generated single-stranded DNA (ssDNA) inL. lactis.A possible single-stranded origin of replication was identified by sequence analysis of pWC1 and by comparing levels of ssDNA produced by pCP12 deletion derivatives. The pWC1 replicon may be a useful addition to other replicons currently available for vector construction.

Genome structure of a Lactobacillus plasmid pNMO belonging to the rolling-circle type replicon.

The new small cryptic plasmid pNMO propagating in Lactobacillus casei IAM 1045 was isolated from a naturally occurring Lactobacillus strain N1f. Sequencing analysis revealed that the pNMO genome is a double-stranded DNA of 3522bp, and has two possible open reading frames, rep and pre as well as three other minor ORFs. The predicted rep product (Rep) of 318 amino acids bears close resemblance to other presumed replication proteins (Rep) encoded by the Lactobacillus plasmids such as pLP1, replicating by the rolling-circle (RC) mechanism via single-stranded (SS) DNA intermediates. Upstream rep, pNMO carries two putative replication origins: one is a Rep-mediated nick-site for the plus-strand synthesis (+ori), and the other is a single strand origin (sso) for the minus-strand replication. Preliminary in vivo experiments suggested that pNMO generates SS DNA intermediates in the N1f cells. On the other hand, the putative pNMO pre product (Pre) of 361 amino acids shows extensive similarity to other presumed Pre/Mov (plasmid recombination/ mobilization) proteins encoded by the Lactobacillus plasmids pLB4 and pLAB1000. These results suggested that pNMO replicates via the RC mechanism, and carries the Pre/Mov system.

Effects of the Generation of Single-Stranded DNA on the Maintenance of Plasmid pMV158 and Derivatives in Lactococcus lactis

The effects of the single-strand origins (SSOs) of the broad-host-range streptococcal plasmid pMV158 on (i) the conversion of its single-stranded (ss) DNA replication intermediates to double-stranded (ds) plasmid DNA and (ii) its maintenance were analyzed. pMV158 is distinguished from most other plasmids that replicate by the rolling-circle mechanism by the presence of two single-strand origins of replication, palA and palU. In this paper the results obtained with Lactococcus lactis are presented; complementary studies with Bacillus subtilis are presented in the accompanying paper (Meijer et al. , 1995). In the presence of both SSOs, no ss plasmid DNA was observed in L. lactis. The removal of either palA or palU resulted in the appearance of low amounts of ssDNA. High amounts of ssDNA were detected, however, when both SSOs were deleted. The results indicated that both SSOs were active, albeit that palU was the most effective of the two. In the presence of both SSOs, the plasmid was stably maintained in L. lactis under nonselective growth conditions. Also, the derivatives containing only one of the two SSOs were maintained rather stably. In contrast, the derivative devoid of both SSOs was poorly maintained. It was concluded that, in the absence of a functional SSO, the generation of large amounts of ssDNA drastically reduces the maintenance of pMV158 in L. lactis. The results also showed that the presence of the plasmid-located mob gene, required for conjugative mobilization, was involved neither in the accumulation of ssDNA nor in the maintenance of pMV158.

Effects of the Generation of Single-Stranded DNA on the Maintenance of Plasmid pMV158 and Derivatives in Different Bacillus subtilis Strains

The effects of the single-strand origins (SSOs) of plasmid pMV158 on (i) the conversion of its single-stranded (ss) replication intermediates to double-stranded (ds) plasmid DNA and (ii) its maintenance were analyzed. The rolling-circle plasmid pMV158, which replicates via ssDNA intermediates, contains two single-strand origins (SSOs) of replication, palA and palU. In this paper the results obtained with Bacillus subtilis are described; complementary studies with Lactococcus lactis are presented in the accompanying paper (Meijer et al., 1995). While in L. lactis both SSOs are functional as ssDNA conversion signal, only palU appeared to be active B. subtilis. Similar to the situation in L. lactis, the accumulation of large amounts of ssDNA resulted in a severe decrease in plasmid maintenance in B. subtilis . In the latter bacterium large amounts of ssDNA were only accumulated, however, when plasmids lacking a functional SSO were propagated in RecA mutant strains. In wild-type RecA strains these plasmids accumulated only modest amounts of ssDNA and they were maintained at fairly stable levels. The results suggest that in B. subtilis a RecA-mediated alternative pathway exists for the conversion of ssDNA which can improve plasmid maintenance. In addition to ssDNA accumulation and the antagonizing role of RecA therein, two other plasmid regions were shown to affect pMV158 maintenance in B. subtilis. One was the mob gene region, which had a negative effect on plasmid maintenance, and the other the palA type SSO. Although palA was not functional as an ssDNA conversion signal in B. subtilis, its presence had a positive effect on pMVI58 maintenance.

Localization of the start sites of lagging‐strand replication of rolling‐circle plasmids from Gram‐positive bacteria

A number of small, multicopy plasmids from Gram-positive bacteria replicate by an asymmetric rolling-circle mechanism. Previous studies with several of these plasmids have identified a palindromic sequence, SSOA, that acts as the single-strand origin (SSO) for the replication of the lagging-strand DNA. Although not all the SSOA sequences share DNA sequence homology, they are structurally very similar. We have used an in vitro system to study the lagging-strand replication of several plasmids from Gram-positive bacteria using the SSOA sequences of pT181, pE194 and pSN2 as representative of three different groups of Staphylococcus aureus plasmids. In addition, we have investigated the lagging-strand replication of the pUB110 plasmid that contains an alternative single-strand origin, SSOU. Our results confirm that RNA polymerase is involved in lagging-strand synthesis from both SSOA and SSOU-type lagging-strand origins. Interestingly, while initiation of lagging-strand DNA synthesis of pUB110 occurred predominantly at a single position within SSOU, replication of pT181, pSN2 and pE194 plasmids initiated at multiple positions from SSOA.

Structural and functional analysis of the single-strand origin of replication from the lactococcal plasmid pWV01.

The single-strand origin (SSO) of the rolling-circle (RC), broad-host-range lactococcal plasmid pWVO1 was functionally characterized. The activity of this SSO in the conversion of single-stranded DNA to double-stranded DNA was tested both in vivo and in vitro. In addition, the effect of this SSO on plasmid maintenance was determined. The functional pWVO1 SSO comprises a 250 bp region, containing two inverted repeats (IRs). The activity of each IR was tested, separately and in combination, in a plasmid derivative that was otherwise completely devoid of structures that might function as SSO. One of the IRs (IR I) showed some homology with other previously described SSOs of the SSOA type, as well as with the conversion signal of the Escherichia coli phage phi X174. This IR was shown to have a partial, RNA polymerase-independent activity in complementary strand synthesis, both in vivo and in vitro. The second IR, which had no activity of its own, was required for full SSO activity, both in vivo and in vitro. The conversion of single-stranded DNA to the double-stranded form by the complete SSO was only partly sensitive to inhibition by rifampicin, indicating the existence of an RNA polymerase-independent pathway for this event. The results suggest that the pWVO1 SSO can be activated by two different routes: an RNA polymerase-dependent one (requiring the entire SSO), and an RNA polymerase-independent one (requiring only IR I).

Determination of the single strand origin of Shigella sonnei plasmid pKYM

The Shigella sonnei plasmid pKYM replicates by a rolling-circle mechanism in Escherichia coli. A 571 nucleotides HincII restriction fragment of the pKYM DNA harbors two potential hairpin loops (I and II). We cloned the fragment into a - ori defective M13 vector phage, M13Δ lac183. The chimera phage, MDKY5, showed a larger plaque size, and increased phage yield and rate of progeny replicative form DNA (RF) synthesis. Rifampicin reduced rate of conversion of the single- to double-stranded RF DNA. In addition, we introduced nucleotide deletions within the cloned pKYM DNA, by Bal31 nuclease digestion. Each of the deletion mutants thus constructed was lacking in a sequence containing the hairpin loops and formed smaller plaques. The in vivo analyses revealed that a 136 nucleotides sequence containing the two hairpins I and II is the pKYM minus origin for complementary strand synthesis (single strand origin, referred to as SSO) and harbors a recognition site(s) by host E. coli RNA polymerase, for primer RNA synthesis. Moreover, we found a 24 nt sequence, upstream of the SSO domain having 83% homology to the recombination site A (RS A) which functions in plasmid sitespecific recombination and/or transfer.

Determination of the origin cleavage and joining domain of geminivirus Rep proteins.

Replication of the single-stranded DNA genome of plant geminiviruses follows a rolling circle mechanism. It strictly depends on a 'rolling circle replication initiator protein', the M(r) 41 kDa viral Rep protein, encoded by the C1 or AC1 genes. Using wheat dwarf virus (WDV) and tomato yellow leaf curl virus (TYLCV) as examples, we show that not only the full-size Rep proteins, but also a putative 30 kDa translation product of WDV open reading frame C1-N as well as an artificially shortened 24 kDa Rep of TYLCV, cleave and join single-stranded origin DNA in vitro. Thus the pivotal origin recognition and processing activities of geminivirus Rep proteins must be mediated by the amino-terminal domain of Rep.

Complete Nucleotide Sequence of the Bacillus thuringiensis subsp. israelensis Plasmid pTX14-3 and Its Correlation with Biological Properties

The complete nucleotide sequence of the plasmid pTX14-3 from Bacillus thuringiensis subsp. israelensis has been determined. The circular DNA molecule was 7649 bp and had a G + C content of 35.1%. Twenty-two open reading frames larger than 50 codons were identified. Ten of these open reading frames are suggested to be protein coding regions. The existence of the polypeptides encoded by the mob14-3 and rep14-3 genes were verified by maxi-cells analysis in Escherichia coli. Even though the rep14-3 gene was expressed in E. coli the plasmid pTX14-3 was unable to replicate in this bacterium. The minimal region of the plasmid pTX14-3 required for replication in B. thuringiensis was identified. Potential secondary structures upstream of the rep14-3 gene indicated regulation by antisense RNA and transcription attenuation. Extensive sequence homology with the B. thuringiensis subsp. thuringiensis plasmid pG12 was found in the last part of the mob14-3 gene, downstream of the rep14-3 gene, and in the region containing the single-strand origin of replication (i.e., the minus origin) of pTX14-3. A sequence of 700 bp containing multiple direct repeats was found in an ORF encoding a glycine and proline rich protein of 35.9 kDa. 1.2 kbp upstream and 0.1 kbp downstream of this ORF was found a large direct repeat of 230 bp (87% identity). The region between this direct repeat was often spontaneously deleted from plasmid derivatives containing the entire pTX14-3.

Replication of the promiscuous plasmid pLSI: a region encompassing the minus origin of replication is associated with stable plasmid inheritance

Deletion of a region of the promiscuous plasmid pLS1 encompassing the initiation signals for the synthesis of the plasmid lagging strand led to plasmid instability in Streptococcus pneumoniae and Bacillus subtilis. This defect could not be alleviated by increasing the number of copies (measured as double-stranded plasmid DNA) to levels similar to those of the wild-type plasmid pLS1. Our results indicate that in the vicinity of, or associated with the single-stranded origin region of pLS1 there is a plasmid component involved in its stable inheritance. Homology was found between the DNA gyrase binding site within the par region of plasmid pSC101 and the pLS1 specific recombination site RSB.

Fine Mapping and DNA Sequence of Replication Functions of Bacillus thuringiensis Plasmid pTX14-3

pTX14-3 is a 7.5-kb cryptic plasmid isolated from a Bacillus thuringiensis subspecies israelensis strain. Like many other small plasmids in gram-positive bacteria, pTX14-3 replicates via a single-stranded DNA intermediate. The nucleotide sequence of the replication region was determined and an open reading frame of 636 base pairs encoding a protein necessary for plasmid replication was identified by deletion analysis. No significant homology was found between this open reading frame and those encoding replication proteins identified on other plasmids isolated from gram-positive bacteria, nor could we find any homology to plus origins from other single-stranded DNA plasmids. Consequently, it seems that the replicon of pTX14-3 belongs to a new family of replicons in the group of single-stranded DNA plasmids. The sequence of the single-strand origin (i.e., the minus origin) responsible for the conversion of single-stranded plasmid DNA to double-stranded plasmid DNA was also determined. A partial homology between the minus origin of pTX14-3 and the Bacillus subtilis plasmid pBAA1 was identified. A previously identified locus that suppresses formation of high molecular weight multimers was also minimized and sequenced.

Cell cycle regulated phosphorylation of RPA-32 occurs within the replication initiation complex.

The transition from G1 to S phase of the cell cycle may be regulated by modification of proteins which are essential for initiating DNA replication. One of the first events during initiation is to unwind the origin DNA and this requires a single-stranded DNA binding protein. RPA, a highly conserved multi-subunit single-stranded DNA binding protein, was first identified as a cellular protein necessary for the initiation of SV40 DNA replication. The 32 kDa subunit of RPA has been shown to be phosphorylated at the start of S phase. Using SV40 replication as a model, we have reproduced in vitro the S phase-dependent phosphorylation of RPA-32 and show that it occurs specifically within the replication initiation complex. Phosphorylated RPA-32 is predominantly associated with DNA. Phosphorylation is not a pre-requisite for association with DNA, but occurs after RPA binds to single-stranded DNA formed at the origin during the initiation phase. The protein kinase(s) which phosphorylates RPA-32 is present at all stages of the cell cycle but RPA-32 does not bind to the SV40 origin or become phosphorylated in extracts from G1 cells. Therefore, the cell cycle-dependent phosphorylation of RPA-32 may be regulated by its binding to single-stranded origin DNA during replication initiation.

Enzymes of Human Herpesviruses

The properties of human herpesvirus-encoded enzymes are reviewed and the importance of sequence analysis of viral genomes as well as the experiments on characteristics of enzymes isolated from infected cell cultures are emphasized. The following enzymes are described in detail: DNA replication complex consisting of DNA polymerase, DNA helicase-primase, single-stranded DNA binding protein and origin binding protein, further thymidine kinase, ribonucleotide reductase, deoxyuridine triphosphatase as well as uracil-DNA-glycosylase, deoxyribonuclease and protein kinase. The importance of these enzymes from the point of view of antiviral chemotherapy is discussed.