Initiation Sites For DNA Synthesis Research Articles

DNA-Binding Proteins Regulating pIP501 Transfer and Replication.

pIP501 is a Gram-positive broad-host-range model plasmid intensively used for studying plasmid replication and conjugative transfer. It is a multiple antibiotic resistance plasmid frequently detected in clinical Enterococcus faecalis and Enterococcus faecium strains. Replication of pIP501 proceeds unidirectionally by a theta mechanism. The minimal replicon of pIP501 is composed of the repR gene encoding the essential rate-limiting replication initiator protein RepR and the origin of replication, oriR, located downstream of repR. RepR is similar to RepE of related streptococcal plasmid pAMβ1, which has been shown to possess RNase activity cleaving free RNA molecules in close proximity of the initiation site of DNA synthesis. Replication of pIP501 is controlled by the concerted action of a small protein, CopR, and an antisense RNA, RNAIII. CopR has a dual function: It acts as transcriptional repressor at the repR promoter and, in addition, prevents convergent transcription of RNAIII and repR mRNA (RNAII), which indirectly increases RNAIII synthesis. CopR binds asymmetrically as a dimer at two consecutive binding sites upstream of and overlapping with the repR promoter. RNAIII induces transcriptional attenuation within the leader region of the repR mRNA (RNAII). Deletion of either control component causes a 10- to 20-fold increase of plasmid copy number, while simultaneous deletions have no additional effect. Conjugative transfer of pIP501 depends on a type IV secretion system (T4SS) encoded in a single operon. Its transfer host-range is considerably broad, as it has been transferred to virtually all Gram-positive bacteria including Streptomyces and even the Gram-negative Escherichia coli. Expression of the 15 genes encoding the T4SS is tightly controlled by binding of the relaxase TraA, the transfer initiator protein, to the operon promoter overlapping with the origin of transfer (oriT). The T4SS operon encodes the DNA-binding proteins TraJ (VirD4-like coupling protein) and the VirB4-like ATPase, TraE. Both proteins are actively involved in conjugative DNA transport. Moreover, the operon encodes TraN, a small cytoplasmic protein, whose specific binding to a sequence upstream of the oriT nic-site was demonstrated. TraN seems to be an effective repressor of pIP501 transfer, as conjugative transfer rates were significantly increased in an E. faecalis pIP501ΔtraN mutant.

An end for mismatch repair

DNA mismatch repair (MMR) is an important postreplicative repair mechanism that removes DNA polymerization errors and is responsible for increasing the fidelity of genome replication by orders of magnitude. Defects in MMR lead to the most common form of hereditary colon cancer and a variety of sporadic cancers (1–3). One of the main features of MMR is its ability to distinguish old and new DNA strands, but when a mismatch is found, how does MMR determine which sequence is the correct one? One of the first papers to describe MMR suggested that the MMR repair complex could have a special relationship with the replication apparatus (4), and that idea seems to be a central feature of strand discrimination in eukaryotes. In a study published in PNAS, Nick McElhinny et al. (5) provide evidence that the close relationship between MMR and replication leads to preferential repair of mismatches near initiation sites for DNA synthesis.

Chemical footprinting of structural and functional elements of dhfr oriβ during the CHOC 400 cell cycle

Oriβ, an origin of replication 3′ to Chinese hamster dihydrofolate reductase ( dhfr) gene, contains several sequence elements that function as components of a chromosomal replicator. Here we have examined sensitivity to KMnO 4 in vitro and in living cells of three regions within dhfr oriβ which contribute to replicator function: the origin of bidirectional DNA replication (OBR) that serves as an initiation site for DNA synthesis, a stably bent DNA region that binds activator protein one (AP-1) and RIP60 in vitro, and an AT-rich region that contains a dA/dT 23 dinucleotide repeat that has properties of a DNA unwinding element. The in vitro patterns of KMnO 4 modification in linear plasmid differed from that in supercoiled plasmid most prominently in the dA/dT 23 repeat, with evidence of palindrome extrusion in supercoiled plasmid. Although palindrome extrusion was not detected in genomic DNA during the cell cycle, the pattern of genomic DNA modification within the dA/dT 23 repeat differed substantially from that of either linear or plasmid DNA in vitro. An AT-rich region that borders the dA/dT repeat was also highly sensitive to modification by KMnO 4 in cells. Within the bent DNA region, the patterns of chemical modification of both the AP-1 and RIP60 sites differed between plasmid and genomic DNA, and minor differences in the in vitro and cellular modification patterns also were observed for the OBR. Nonetheless, there was little evidence of cell cycle-specific modifications in any sequence examined. These studies suggest that sequences within dhfr oriβ adopt specific conformations in cells, with the most prominent changes in the AT-rich region associated with the dA/dT 23 repeat and DNA unwinding.

Identification of short 'eukaryotic' Okazaki fragments synthesized from a prokaryotic replication origin.

Although archaeal genomes encode proteins similar to eukaryotic replication factors, the hyperthermophilic archaeon Pyrococcus abyssi replicates its circular chromosome at a high rate from a single origin (oriC) as in Bacteria. In further elucidating the mechanism of archaeal DNA replication, we have studied the elongation step of DNA replication in vivo. We have detected, in two main archaeal phyla, short RNA-primed replication intermediates whose structure and length are very similar to those of eukaryotic Okazaki fragments. Mapping of replication initiation points further showed that discontinuous DNA replication in P. abyssi starts at a well-defined site within the oriC recently identified in this hyperthermophile. Short Okazaki fragments and a high replication speed imply a very efficient turnover of Okazaki fragments in Archaea. Archaea therefore have a unique replication system showing mechanistic similarities to both Bacteria and Eukarya.

Chromosomal ARS1 Has a Single Leading Strand Start Site

Initiation sites for DNA synthesis in the chromosomal autonomously replicating sequence (ARS)1 of Saccharomyces cerevisiae were detected at the nucleotide level. The transition from discontinuous to continuous synthesis defines the origin of bidirectional replication (OBR), which mapped adjacent to the origin recognition complex binding site. To ascertain which sites represented starts for leading or lagging strands, we characterized DNA replication from ARS1 in a cdc9 (DNA ligase I) mutant, defective for joining Okazaki fragments. Leading strand synthesis in ARS1 initiated at only a single site, the OBR. Thus, replication in S. cerevisiae is not initiated stochastically by choosing one out of multiple possible sites but, rather, is a highly regulated process with one precise start point.

Replication terminus for DNA polymerase I during initiation of pAM beta 1 replication: role of the plasmid-encoded resolution system.

Replication of plasmid pAM beta 1 is initiated by DNA polymerase I (Pol I) and completed by DNA polymerase III holoenzyme contained in the replisome machinery. In this study we report that initiation of DNA replication generates D-loop structures containing the nascent leading strand paired to its template, and that D-loop extension is arrested approximately 230 bp from the initiation site of DNA synthesis in the presence of the plasmid-encoded resolvase. In vitro and in vivo data suggest that this arrest is caused by a collision between Pol I and the resolvase bound to its target. As the arrested D-loop replication intermediates carry a single-stranded primosome-assembly site, we hypothesize that the biological role of the replication arrest is to limit the region replicated by Pol I and to promote the replacement of Pol I by the replisome in order to initiate concerted synthesis of the leading and lagging strands.

Identification of a signal necessary for initiation of reverse transcription of the hepadnavirus genome

Reverse transcription of the hepadnavirus genome initiates near the 3' end of the RNA template and has previously been shown to depend on sequences flanking the initiation site for DNA synthesis (C. Seeger and J. Maragos, J. Virol. 64:16-23, 1990). DNA synthesis leads to the covalent attachment of a protein to the 5' end of minus-strand DNA, and it is generally believed that this protein serves as the primer for reverse transcription. To examine priming in more detail, we have carried out a detailed genetic analysis of the nucleotide sequences at the origin of minus-strand DNA synthesis characterized in our earlier study. This mutational analysis has led to the identification of a short, four-nucleotide-long sequence as the signal for initiation of reverse transcription. This signal is a UUUC sequence motif flanking the position of the 5' end of minus-strand DNA, which alone is not sufficient for DNA synthesis, indicating that positional effects are also important to specify the origin of DNA synthesis.

Mapping Eukaryotic Replication OriginsIn Vivoby Size Analysis of Purified Nascent DNA Strands

A simple and efficient method for the mapping of eukaryotic replication origins was tested. The method is based on differential labeling of newly synthesized DNA with BrdUrd and subsequent separation of heavy nascent strands from parental DNA by conventional alkaline sucrose and neutral CsCl isopycnic gradient centrifugation. Purified nascent DNA is then size-fractionated on alkaline agarose gels and analyzed by sequential hybridization to specific probes of known location on the DNA segment of interest. Evaluation of the hybridization results allows: (i) determination of the direction of replication fork movement and (ii) location of the initiation site of DNA synthesis. Taking SV40 and polyoma virus as model systems, we demonstrate the feasibility of this procedure. It applicability to the location of chromosomal replication origins is discussed.

Deletion of sites for initiation of DNA synthesis in the origin of broad host-range plasmid R1162

The origin of replication of the broad host-range plasmid R1162 contains two, oppositely facing initiation sites for DNA synthesis. Either of these sites can be deleted from an R1162 plasmid derivative. However, the resulting plasmids are unstable, maintained at a lower copy-number in the cell, and form dimers and other recombinants that are required for propagation of the plasmid. In vitro, a derivative lacking one initiation site is deficient in synthesis of the strand normally initiated from that site. The properties of the intact origin are restored if it contains two oppositely facing sites; one initiation site may substitute for the other, and each site need not be in its original orientation. Overall, the results suggest that synthesis of each strand of R1162 DNA is initiated at a single site, and that there is no efficient system for initiation of lagging strand synthesis during transit of the replication forks.

Identification and characterization of the woodchuck hepatitis virus origin of DNA replication

Replication of the woodchuck hepatitis virus (WHV) genome requires the synthesis of minus-strand DNA from an RNA template, the pregenome, by reverse transcription. During this reaction, the 5' end of minus-strand DNA becomes covalently linked to a protein. The position of the 5' end of minus-strand DNA was identified previously, but the initiation site for DNA synthesis on pregenomic RNA remained ambiguous because of a sequence repetition at the termini of the RNA template for reverse transcription. Employing a recently designed expression vector for the production of infectious WHV, we localized the origin of minus-strand DNA synthesis to the 3' end of pregenomic RNA. In addition, we identified the nucleotide sequences on pregenomic RNA that provide the signal for the initiation of reverse transcription. Removal of this signal sequence from pregenomic RNA abolished minus-strand DNA synthesis. Insertion of a DNA oligomer bearing this signal sequence at the 3' end of pregenomic RNA restored the production of minus-strand DNA joined to protein. Our results support a model in which protein is the primer for reverse transcription of minus-strand DNA of WHV.

The distribution and properties of RNA primed initiation sites of DNA synthesis at the replication origin ofEscherichia colichromosome

RNA-linked DNA molecules were obtained from E. coli dnaCts cells synchronously initiating a new round of chromosome replication. The deoxynucleotides at the transition from primer RNA to DNA were 32P-labeled, and their positions were located on the nucleotide sequence of 1.4 kb genomic region (position -906 to +493) including the oriC and its leftside flanking region. In the r-strand (the counterclockwise strand), many strong transition sites were mapped in the left half portion of the oriC and a few weak sites in the left outside region. In the 1-strand (the clockwise strand), no transition sites were found inside the oriC but many weak sites were found in the left outside region. The results support the initiation mechanism in which the first leading strand synthesis starts with the r-strand counterclockwise from the oriC that is followed by the 1-strand synthesis on the displaced template strand on the left of oriC. Primer RNA molecules attached to the strong r-strand transition sites were only a few residues in length. Properties of the transition sites were discussed.

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.

Initiation of SV40 DNA replication in vivo: Location and structure of 5′ ends of DNA synthesized in the ori region

Initiation sites for DNA synthesis were located at the resolution of single nucleotides in and about the genetically defined origin of replication ( ori) in replicating SV40 DNA purified from virus-infected cells. About 50% of the DNA chains contained an oligoribonucleotide of six to nine residues covalently attached to their 5′ ends. Although the RNA-DNA linkage varied, the putative RNA primer began predominantly with rA. The data reveal that initiation of DNA synthesis is promoted at a number of DNA sequences that are asymmetrically arranged with respect to ori: 5′ ends of nascent DNA are located at several sites within ori, but only on the strand that also serves as the template for early mRNA, while 5′ ends of nascent DNA with the opposite orientation are located only outside ori on its early gene side. This clear transition between discontinuous (initiation sites) and continuous (no initiation sites) DNA synthesis defines the origin of bidirectional replication at nucleotides 5210–5211 and demonstrates that discontinuous synthesis occurs predominantly on the retrograde arms of replication forks. Furthermore, it appears that the first nascent DNA chain is initiated within ori by the same mechanism used to initiate nascent DNA (“Okazaki fragments”) throughout the genome.

Distribution of a platinum anti-tumour drug in HeLa cells by analytical electron microscopy

The distribution of Pt in HeLa cell sections after cell treatment with cis-Pt(NH 3) 2Cl 2 dissolved in dimethylsulphoxide was probed by analytical electron microscopy. Primary targets were the nucleolus and the inner side of the nuclear double membrane. Even after solvolysis in dimethylsulphoxide the drug reached similar sites. It is suggested that cell death may be due to Pt inhibition at the initiation sites of DNA synthesis.

Construction of a viable SV40 variant containing two functional origins of DNA replication

Viable variants of simian virus 40 (SV40) have been constructed which contain two functional origins of DNA replication (Or). The variants were made by introducing, at 0.175 on the SV40 map, a segment of DNA containing the viral Or. Two types of experiments demonstrate that the second Or is functional. First, the distribution of radioactivity in pulse-labeled SV40 (I) DNA is dramatically altered in the variants when compared with the parental virus. Second, electron microscopic examination of viral replicative intermediates indicates that while there is one initiation site for DNA synthesis in the parental genome, there are two sites in the variant. It was possible to introduce a deletion which inactivated the original Or at 0.67 map units in this variant. The resulting mutant could be propagated, and its DNA replication originated at the site of the newly inserted Or.

Nucleotide sequence of Rous sarcoma virus RNA at the initiation site of DNA synthesis: The 102nd nucleotide is U

The T 1 oligonucleotide in the genome Rous sarcoma virus (RSV) that corresponds to the initiation site of DNA synthesis in vitro was identified by hybridization of genome RNA with RSV strong stop DNA (the initial 101-nucleotide long fragment synthesized in endogenous reactions) and partially sequenced. The sequence of (C 2, U 2) A-U-U-U-G found corresponds to the d(A-A-T-G-A-A-G) sequence at the 5′ end of the DNA product plus the CA-OH sequence at the 3′ end of the tRNA Trp primer. Therefore the nucleotide opposite the terminal A of the primer is the complementary U. Furthermore, no internal repetition of more than 30 nucleotides of the 5′ sequence could be detected.

Fiber autoradiography of replicating yeast DNA

The replication of yeast nuclear DNA was examined by DNA fiber autoradiography. We found that yeast chromosomes contain multiple initiation sites for DNA synthesis. DNA is replicated bidirectionally from these sites at a rate of approx. 0.7 μm/min/replication fork at 24 °C. In these respects DNA replication in this simple eucaryote is similar to DNA replication in mammalian cells.

Nature of the base sequence conserved in the mitochondrial DNA of a low-density petite

1. 1. We have previously shown that the mtDNA of the ethidium-induced cytoplasmic petite mutant RD1A of the yeast Saccharomyces cerevisiae consists of perfect tandem repetitions of a very (A+T)-rich sequence of < 300 base pairs. 2. 2. The complementary strands of RD1A mtDNA were separated in alkaline CsCl and used for DNA · DNA hybridization with wild-type mtDNA and other petite mtDNAs. Hybridization was analysed either by standard filter techniques or with purified S 1 nuclease from Aspergillus oryzae, which is specific for single-stranded DNA. The hybridization plateau of 0.3 % indicates that one copy of the repeating sequence of RD1A mtDNA is present in wild-type mtDNA, but the presence of up to 3 copies could not be excluded. 3. 3. The T m of the heteroduplex of wild-type and RD1A mtDNA on hydroxylapatite is 6 °C lower than that of the RD1A mtDNA homoduplex. This shows that some miscopying must have occurred during the initial mutagenic event that gave rise to RD1A mtDNA. The RD1A mtDNA sequence is also present in mtDNA of Saccharomyces carlsbergensis but in this case the T m of the heteroduplex is 13 °C below that of the homoduplex. 4. 4. No sequence homology was detected between mtDNA of RD1A and the repetitive (A+T)-rich mtDNAs of two other petite mutants, either in DNA · DNA hybridization or in DNA · RNA hybridization using complementary RNA made with RNA polymerase of Escherichia coli. This lack of homology was confirmed by the fingerprints of the complementary RNAs of the three DNAs. These results indicate that the initiation site for DNA synthesis does not form part of the repeating sequence of these mtDNAs. 5. 5. The analogy between the repetitive petite mtDNAs and the repetitive nuclear satellite DNAs of animal tissues, is stressed.

Fine structure of interphase nuclei

The initiation and replication sites of DNA synthesis in the plasmodial nuclei of Physarum polycephalum were studied with electron microscopic autoradiography. By using both thin sectioning and whole mount techniques, it was shown that the dense chromatin masses in the nucleus consisted of predominantly elementary chromatin-like fibrils, approximately 300 A in diameter while the electron transparent region in the nucleus consisted of predominantly finer fibrils, less than 100 A in diameter. With electron microscopic autoradiography it was found that (1) the initiation sites of DNA synthesis were definitely in the boundary regions between the dense chromatin masses and the electron transparent region, (2) the initiation and replication sites of DNA synthesis were definitely not on the nuclear membrane, (3) within a few minutes, replication sites migrated from the initiation sites to the electron transparent region and (4) in this electron transparent region, almost all of the nuclear DNA was synthesized.

Fine Structures of Interphase Nuclei

The initiation and replication sites of DNA synthesis in the plasmodial nuclei of Physarum polycephalum were studied with electron microscopic autoradiography. By using both thin sectioning and whole mount techniques, it was shown that the dense chromatin masses in the nucleus consisted of predominantly elementary chromatin-like fibrils, approximately 300 A in diameter while the electron transparent region in the nucleus consisted of predominantly finer fibrils, less than 100 A in diameter. With electron microscopic autoradiography it was found that (1) the initiation sites of DNA synthesis were definitely in the boundary regions between the dense chromatin masses and the electron transparent region, (2) the initiation and replication sites of DNA synthesis were definitely not on the nuclear membrane, (3) within a few minutes, replication sites migrated from the initiation sites to the electron transparent region and (4) in this electron transparent region, almost all of the nuclear DNA was synthesized.