Biomedical subjects

H Hiasa

Publications and source records attributed to H Hiasa.

33 records · Page 2Linked to original sources

PubMed1994

Tus prevents overreplication of oriC plasmid DNA.

Minichromosome plasmid DNA templates containing oriC and two Ter sites, oriented as they are on the Escherichia coli chromosome, have been used to study the role of Tus in termination of bidirectional replication. In replication reactions reconstituted with purified proteins where it could be demonstrated that each active template was replicating bidirectionally, Tus was required to prevent extensive overreplication. In the presence of Tus, both replication forks terminated DNA synthesis at one or the other Ter site in an apparent stepwise manner. First, the progress of one replication fork was arrested by a properly oriented Tus-Ter complex. Then, either because of steric hindrance resulting from the stalled replication machinery of the first fork or because of the formation of a branched DNA structure, the progression of the second opposing fork was halted at the same site on the DNA template. In the absence of Tus, overreplication required DNA ligase and arose via a template strand-switching mechanism. Thus, the role of Tus in E. coli is more likely to prevent overreplication rather than to ensure accurate termination.

Bacterial Proteins↗

PubMed1994

Fis cannot support oriC DNA replication in vitro.

The effects of the histone-like proteins, HU, IHF, and Fis on DNA replication have been examined in vitro using the oriC and pBR322 DNA replication systems. In the oriC system, although low concentrations of HU and IHF stimulated DNA replication, no stimulation was observed upon the addition of Fis. On the contrary, high concentrations of Fis inhibited oriC DNA replication completely. The inhibitory effect of Fis could be overcome when HU, IHF, or RNA polymerase were also present in the reaction mixtures. Unwinding of duplex DNA at the origin correlated with the elevated level of DNA replication. In contrast, only inhibition, rather than stimulation, was observed when these proteins were added to the pBR322 DNA replication system. Thus, the observed dependence on Fis of oriC DNA replication in vivo cannot be attributed to a positive effect on localized DNA unwinding.

Bacterial Proteins↗

PubMed1994

Topoisomerase IV can support oriC DNA replication in vitro.

Escherichia coli has two type II topoisomerases, DNA gyrase and topoisomerase IV (Topo IV). Topo IV is required for the decatenation of the linked daughter chromosomes at the terminal stages of DNA replication, whereas gyrase, because of its ability to convert to negative supercoils the positive supercoils generated by replication fork progression in a circular chromosome, is required to support nascent chain elongation. Using an oriC DNA replication system in vitro, we show that Topo IV, which can relax positive supercoils, can also support replication fork progression. This activity is only observed at substoichiometric ratios of Topo IV to template, at higher ratios, the template becomes relaxed and initiation of DNA replication cannot occur. Topo IV was capable of supporting bidirectional DNA replication from oriC, although, unlike the case with gyrase, some templates apparently replicated unidirectionally. This suggests that either gyrase itself or a certain minimum superhelical density is required for proper initiation of DNA replication from oriC.

Cell-Free System↗

PubMed1994

Primase couples leading- and lagging-strand DNA synthesis from oriC.

Coupling of leading- and lagging-strand DNA synthesis at replication forks formed at Escherichia coli oriC has been studied in vitro using a replication system reconstituted with purified proteins. At low concentrations of primase (8 nM), the major replication products were multigenome-length molecules, generated by a rolling circle-type mechanism, and unit-length molecules. Rolling circle DNA replication was inhibited at high concentrations of primase (80 nM) and the major replication products were half-unit-length leading strands and a distinct population of short Okazaki fragments. At low primase concentrations, an asymmetric mode of DNA synthesis occurred. Each strand was made independently and initiation could occur outside of oriC. At high primase concentrations, initiation occurred exclusively at oriC and two coupled replication forks proceeded bidirectionally around the plasmid. Presumably, at low concentrations of primase, DnaB (the replication fork helicase) unwound the plasmid DNA before replication forks could form, leading to initiation at sites other than oriC. On the other hand, high concentrations of primase resulted in successful capture of the helicase leading to the formation at oriC of coupled replication forks capable of coordinated leading- and lagging-strand synthesis.

DNA Primase↗

PubMed1994

Decatenating activity of Escherichia coli DNA gyrase and topoisomerases I and III during oriC and pBR322 DNA replication in vitro.

oriC and pBR322 DNA replication, reconstituted with purified replication proteins, has been used to study the functional activities of Escherichia coli topoisomerase I, DNA gyrase, and topoisomerase III during the final stages of DNA replication. In the oriC system, DNA gyrase-catalyzed decatenation of daughter DNA molecules was very inefficient, whereas topoisomerase III could catalyze complete decatenation. In the pBR322 DNA replication system, almost all the daughter DNA molecules could be decatenated by DNA gyrase alone in the absence of salt. Decatenation by DNA gyrase in the pBR322 system was completely inhibited, without a concomitant inhibition of DNA synthesis, by the addition of physiological concentrations of salt. Topoisomerase III, however, could decatenate all of the daughter DNA molecules in the pBR322 system, even in the presence of high concentrations of salt. A similar effect could not be observed in the oriC system, because the addition of salt inhibited DNA synthesis. Topoisomerase I was incapable of catalyzing decatenation under any conditions examined in either the oriC or pBR322 replication system. The addition of topoisomerase I to the replication systems resulted only in an inhibition of DNA synthesis.

Adenosine Triphosphate↗

PubMed1994

Comparative analysis of functional and structural features in the primase-dependent priming signals, G sites, from phages and plasmids.

The primase-dependent priming signals, G sites, are directly recognized by the Escherichia coli primase (dnaG gene product) and conduct the synthesis of primer RNAs. In nucleotide sequence and secondary structure, there is no striking resemblance between the phage- and plasmid-derived G sites, except for the limited sequence homology near the start position of primer RNA synthesis. In this study, we analyzed the structure and function of a G site of plasmid R100, G site (R100), and discovered the necessity of the coexistence of two domains (domains I and III), which contains blocks A, B, and C, which are nucleotide sequences highly conserved among the plasmid-derived G sites. However, neither the internal region, domain II, between domains I and III nor the potential secondary structure proposed by Bahk et al. (J. D. Bahk, N. Kioka, H. Sakai, and T. Komano, Plasmid 20:266-270, 1988) is essential for single-stranded DNA initiation activity. Furthermore, chimeric G sites constructed between a G site of phage G4, G site(G4), and G site(R100) maintained significant single-stranded DNA initiation activities. These results strongly suggest that phage- and plasmid-derived G sites have functionally equivalent domains. The primase-dependent priming mechanisms of phage- and plasmid-derived G sites are discussed.

Base Sequence↗

PubMed1992

Differential inhibition of the DNA translocation and DNA unwinding activities of DNA helicases by the Escherichia coli Tus protein.

Binding of the Escherichia coli Tus protein to its cognate nonpalindromic binding site on duplex DNA (a Ter sequence) is sufficient to arrest the progression of replication forks in a Ter orientation-dependent manner in vivo and in vitro. In order to probe the molecular mechanism of this inhibition, we have used a strand displacement assay to investigate the effect of Tus on the DNA helicase activities of DnaB, PriA, UvrD (helicase II), and the phi X-type primosome. When the substrate was a short oligomer hybridized to a circular single-stranded DNA, strand displacement by DnaB, PriA, and the primosome (in both directions), but not UvrD, was blocked by Tus in a polar fashion. However, no inhibition of either DnaB or UvrD was observed when the substrate carried an elongated duplex region. With this elongated substrate, PriA helicase activity was only inhibited partially (by 50%). On the other hand, both the 5'----3' and 3'----5' helicase activities of the primosome were inhibited almost completely by Tus with the elongated substrate. These results suggest that while Tus can inhibit the translocation of some proteins along single-stranded DNA in a polar fashion, this generalized effect is insufficient for the inhibition of bona fide DNA helicase activity.

Bacterial Proteins↗

PubMed1992

Alternative secondary structures in the phage G4 origin of the complementary DNA strand synthesis: effects of NaCl concentration on the bleomycin-DNA interaction.

The effects of NaCl concentration on bleomycin-induced cleavages of single-strand and double-strand DNA fragments containing the phage G4 origin of complementary DNA strand synthesis were investigated. It was found that bleomycin could be used as a reagent to analyze secondary and tertiary structures and subtle changes of DNA structures. The effects of NaCl concentration on cleavages of single-stranded DNA were distinct at every target site, indicating that the diversity of topolotical properties of DNA might change the selectivity of the bleomycin-induced DNA cleavage. These results showed alternative secondary structures within and close to the G4 origin of complementary DNA strand synthesis.

Bacteriophages↗

PubMed1991

Functional division and reconstruction of a plasmid replication origin: molecular dissection of the oriV of the broad-host-range plasmid RSF1010.

Two single-stranded DNA initiation signals (designated ssi) present in the origin of vegetative DNA replication (oriV) of the broad-host-range plasmid RSF1010 are essential for the priming of replication of each complementary DNA strand of this plasmid in Escherichia coli. Each of the RSF1010 ssi signals, ssiA and ssiB, could be replaced by a primosome assembly site from plasmid pACY184 or from bacteriophage phi X174. In these chimeric origins, replication of the strand complementary to that containing the primosome assembly site was no longer dependent on the RSF1010 primase, protein RepB', but required the E. coli primase, DnaG. If both ssiA and ssiB sites of RSF1010 were replaced by primosome assembly sites, protein RepB' was no longer essential for the replication at this origin, whereas proteins RepA and RepC of RSF1010 were still required. These results strongly suggest that the two ssi sites and the RepB' protein actually direct the priming of DNA synthesis in the replication of RSF1010, and the proteins RepA and RepC are involved in the prepriming events--i.e., the opening of the DNA duplex at oriV. It is evident that the origin of RSF1010 can be separated into three functional domains and reconstructed by replacing the ssi sites with heterologous elements.

Base Sequence↗

PubMed1991

Plasmid Co1IB contains an ssi signal close to the replication origin.

Taking advantage of the plaque morphology method, we identified a single-strand initiation (ssi) signal in plasmid pSM32, a mini-Co1Ib plasmid. This ssi signal was situated in the 350-nt HaeIII segment of the 1.8-kb S7 fragment, and located nearly 400 nt downstream of the origin of DNA replication. Introduction of the ssi signal into a mutant of filamentous phage M13 lacking oric resulted in restoration of phage growth and RFI DNA synthesis. Interestingly, DNA homology studies showed that the nucleotide sequence of the ssi signal was extremely homologous with that of the "G4-type" ssi signal in plasmid R100.

Base Sequence↗

PubMed1990

Structural features of the priming signal recognized by primase: mutational analysis of the phage G4 origin of complementary DNA strand synthesis.

45 mutations (insertion, deletion and base substitution) of the G4 Goric were tested for their functional activity in M13 and R199 in vivo. The critical mutants were also assayed for their ability to synthesize pRNA in vitro using SSB and primase. The results demonstrate that the secondary structure and spacing of stem-loops I and III are essential for Goric activity and that the 5'-CTG-3' sequence flanking stem-loop I is essential for initiation of pRNA synthesis.

Bacteriophages↗

PubMed1989

Mutational analysis of the primer RNA template region in the replication origin (oric) of bacteriophage G4: priming signal recognition by Escherichia coli primase.

The primase-dependent phage G4 origin of complementary DNA strand synthesis (G4oric) contains three stable stem-loops (I, II, and III) upstream from the initiation point of primer RNA (pRNA). Site-directed mutagenesis was used to introduce alterations into the nucleotide (nt) sequence of the G4oric pRNA template region. Mutations in stem-loop I, that changed the length of the stem and the sequence of the loop, slightly depressed, but did not abolish, G4oric activity. However, functional G4oric activity was destroyed when the sequence containing the starting position of pRNA synthesis was deleted, or when insertions were introduced between the pRNA starting position (5'-CTG-3') and stem-loop I. Reintroducing a CTG as part of a PstI linker close to stem-loop I, however, resulted in recovery of G4oric functional activity. These results suggest that the specific nt sequence, containing 5'-CTG-3', between nt 3994 and 4007, and also the distance between the starting position of pRNA synthesis and stem-loop I, are essential structural features for G4oric function.

Base Sequence↗

PubMed1989

Distinct functional contributions of three potential secondary structures in the phage G4 origin of complementary DNA strand synthesis.

Three potential secondary structures, stem-loops I, II, and III, are contained in the phage G4 origin of complementary DNA strand synthesis, G4oric, and are believed to be involved in its recognition by dnaG-encoded primase and the synthesis of primer RNA. In a previous publication [Sakai et al., Gene 71 (1988) 323-330], we suggested that base pairing between the loops of stem-loops I, and II, and/or II and III, might play a role in G4oric function. To test this hypothesis, site-directed mutagenesis was used to construct mutants which carried base substitutions in loops I, II and III that destroyed possible interloop base pairing. These mutations, however, did not seriously affect G4oric activity. This indicates that base pairing between the loops is not essential for G4oric functional activity, and also that base substitutions which do not affect the secondary structure of stem-loops I, II and III, do not affect G4oric activity. To complete an analysis of the effects of altering the structure of the G4oric stem-loops, insertions were made into stem-loop III. In contrast to stem-loops I and II, all insertions into stem-loop III destroyed in vivo G4oric activity.

Base Sequence↗

PubMed1989

Identification of single-strand initiation signals in the terC region of the Escherichia coli chromosome.

On the basis of clear-plaque formation, we detected initiation signals in the terC region of the Escherichia coli chromosome. At least two single-strand initiation signals were identified from the terC region. The nucleotide sequences of these two signals were determined. Sequence homologies, variations of the consensus of n' protein recognition sites, 5'-GAAGCGG-3', were found within these signals. A novel conserved sequence was also found within these signals. Their initiation activities were measured both by the infection growth assay and by the ability to convert the single-stranded DNA to the duplex replicative form DNA in vivo.

Bacteriophage phi X 174↗

PubMed1988

Role of the potential secondary structures in phage G4 origin of complementary DNA strand synthesis.

Phage G4 origin of complementary DNA strand synthesis (oric) consists of three stable stem-loop structures (I, II, and III). Mutant oric sequences with alterations in the structure of stem-loop II, stem-loop III, and the stem-loop II-III spacer region have been constructed and cloned into the filamentous phage vectors to assay their functional activity. Changes in the lowermost GC base pair in the stem of stem-loop III, in the 9-bp spacer region between the stems of stem-loops II and III, and in the loop of stem-loop II, impair or abolish in vivo oric function. The results suggest that recognition sequences for dnaG primase must be present in the loop of stem-loop II, and in the spacer region between the stems of stem-loops II and III.

Bacteriophages↗