scholarly journals Mapping in vivo topoisomerase I sites on simian virus 40 DNA: asymmetric distribution of sites on replicating molecules.

1989 ◽  
Vol 9 (2) ◽  
pp. 541-550 ◽  
Author(s):  
S E Porter ◽  
J J Champoux
Keyword(s):  
Dna Replication ◽  
Topoisomerase I ◽  
Consensus Sequence ◽  
Simian Virus 40 ◽  
Unknown Origin ◽  
Simian Virus ◽  
Asymmetric Distribution ◽  
Potential Break

Complexes between simian virus 40 DNA and topoisomerase I (topo I) were isolated from infected cells treated with camptothecin. The topo I break sites were precisely mapped by primer extension from defined oligonucleotides. Of the 56 sites, 40 conform to the in vitro consensus sequence previously determined for topo I. The remaining 16 sites have an unknown origin and were detectable even in the absence of camptothecin. Only 11% of the potential break sites were actually broken in vivo. In the regions mapped, the pattern of break sites was asymmetric. Most notable are the clustering of sites near the terminus for DNA replication and the confinement of sites to the strand that is the template for discontinuous DNA synthesis. These asymmetries could reflect the role of topo I in simian virus 40 DNA replication and suggest that topo I action is coordinated spatially with that of the replication complex.

Mapping in vivo topoisomerase I sites on simian virus 40 DNA: asymmetric distribution of sites on replicating molecules

1989 ◽  
Vol 9 (2) ◽  
pp. 541-550
Author(s):  
S E Porter ◽  
J J Champoux
Keyword(s):  
Dna Replication ◽  
Topoisomerase I ◽  
Consensus Sequence ◽  
Simian Virus 40 ◽  
Unknown Origin ◽  
Simian Virus ◽  
Asymmetric Distribution ◽  
Potential Break

Complexes between simian virus 40 DNA and topoisomerase I (topo I) were isolated from infected cells treated with camptothecin. The topo I break sites were precisely mapped by primer extension from defined oligonucleotides. Of the 56 sites, 40 conform to the in vitro consensus sequence previously determined for topo I. The remaining 16 sites have an unknown origin and were detectable even in the absence of camptothecin. Only 11% of the potential break sites were actually broken in vivo. In the regions mapped, the pattern of break sites was asymmetric. Most notable are the clustering of sites near the terminus for DNA replication and the confinement of sites to the strand that is the template for discontinuous DNA synthesis. These asymmetries could reflect the role of topo I in simian virus 40 DNA replication and suggest that topo I action is coordinated spatially with that of the replication complex.

scholarly journals Autonomous replicating sequences from mouse cells which can replicate in mouse cells in vivo and in vitro.

1987 ◽  
Vol 7 (1) ◽  
pp. 1-6 ◽  
Author(s):  
H Ariga ◽  
T Itani ◽  
S M Iguchi-Ariga
Keyword(s):  
Dna Replication ◽  
Dna Sequences ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Copy Numbers ◽  
Mouse Cells ◽  
Simplex Virus

We have already reported that the cloned mouse DNA fragment (pMU65) could replicate in a simian virus 40 T antigen-dependent system in vivo and in vitro (H. Ariga, Z. Tsuchihashi, M. Naruto, and M. Yamada, Mol. Cell. Biol. 5:563-568, 1985). The plasmid p65-tk, containing the thymidine kinase (tk) gene of herpes simplex virus and the BglII-EcoRI region of pMU65 homologous to the simian virus 40 origin of DNA replication, was constructed. The p65-tk persisted episomally in tk+ transformants after the transfection of p65-tk into mouse FM3Atk- cells. The copy numbers of p65-tk in FM3Atk+ cells were 100 to 200 copies per cell. Furthermore, the p65-tk replicated semiconservatively, and the initiation of DNA replication started from the mouse DNA sequences when the replicating activity of p65-tk was tested in the in vitro DNA replication system developed from the FM3A cells. These results show that a 2.5-kilobase fragment of mouse DNA contains the autonomously replicating sequences.

scholarly journals Mutational Analysis of Simian Virus 40 T-Antigen Primosome Activities in Viral DNA Replication

2002 ◽  
Vol 76 (10) ◽  
pp. 5121-5130 ◽  
Author(s):  
Robert D. Ott ◽  
Yingda Wang ◽  
Ellen Fanning
Keyword(s):  
Dna Replication ◽  
Topoisomerase I ◽  
Mutational Analysis ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Dna Helicase ◽  
T Antigen ◽  
Helicase Domain ◽  
In Vitro Synthesis

ABSTRACT The recruitment of DNA polymerase α-primase (pol-prim) is a crucial step in the establishment of a functional replication complex in eukaryotic cells, but the mechanism of pol-prim loading and the composition of the eukaryotic primosome are poorly understood. In the model system for simian virus 40 (SV40) DNA replication in vitro, synthesis of RNA primers at the origin of replication requires only the viral tumor (T) antigen, replication protein A (RPA), pol-prim, and topoisomerase I. On RPA-coated single-stranded DNA (ssDNA), T antigen alone mediates priming by pol-prim, constituting a relatively simple primosome. T-antigen activities proposed to participate in its primosome function include DNA helicase and protein-protein interactions with RPA and pol-prim. To test the role of these activities of T antigen in mediating priming by pol-prim, three replication-defective T antigens with mutations in the ATPase or helicase domain have been characterized. All three mutant proteins interacted physically and functionally with RPA and pol-prim and bound ssDNA, and two of them displayed some helicase activity. However, only one of these, 5030, mediated primer synthesis and elongation by pol-prim on RPA-coated ssDNA. The results suggest that a novel activity, present in 5030 T antigen and absent in the other two mutants, is required for T-antigen primosome function.

scholarly journals Topoisomerase I Associates Specifically with Simian Virus 40 Large-T-Antigen Double Hexamer–Origin Complexes

2000 ◽  
Vol 74 (11) ◽  
pp. 5224-5232 ◽  
Author(s):  
Dahai Gai ◽  
Rupa Roy ◽  
Chunxiao Wu ◽  
Daniel T. Simmons
Keyword(s):  
Dna Replication ◽  
Topoisomerase I ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Replication Initiation ◽  
Initiation Complex ◽  
Initiation Of Replication ◽  
Sv40 Origin

ABSTRACT Topoisomerase I (topo I) is required for releasing torsional stress during simian virus 40 (SV40) DNA replication. Recently, it has been demonstrated that topo I participates in initiation of replication as well as in elongation. Although T antigen and topo I can bind to one another in vitro, there is no direct evidence that topo I is a component of the replication initiation complex. We demonstrate in this report that topo I associates with T-antigen double hexamers bound to SV40 origin DNA (TDH) but not to single hexamers. This association has the same nucleotide and DNA requirements as those for the formation of double hexamers on DNA. Interestingly, topo I prefers to bind to fully formed TDH complexes over other oligomerized forms of T antigen associated with the origin. High ratios of topo I to origin DNA destabilize TDH. The partial unwinding of a small-circular-DNA substrate is dependent on the presence of both T antigen and topo I but is inhibited at high topo I concentrations. Competition experiments with a topo I-binding fragment of T antigen indicate that an interaction between T antigen and topo I occurs during the unwinding reaction. We propose that topo I is recruited to the initiation complex after the assembly of TDH and before unwinding to facilitate DNA replication.

DNA primase-DNA polymerase alpha from simian cells: sequence specificity of initiation sites on simian virus 40 DNA

1985 ◽  
Vol 5 (5) ◽  
pp. 1170-1183
Author(s):  
M Yamaguchi ◽  
E A Hendrickson ◽  
M L DePamphilis
Keyword(s):  
Dna Polymerase ◽  
Consensus Sequence ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Sequence Specificity ◽  
Dna Polymerase Alpha ◽  
Dna Primase ◽  
Template Sequence

Unique single-stranded regions of simian virus 40 DNA, phage M13 virion DNA, and several homopolymers were used as templates for the synthesis of (p)ppRNA-DNA chains by CV-1 cell DNA primase-DNA polymerase alpha. Intact RNA primers, specifically labeled with an RNA capping enzyme, were typically 6 to 8 ribonucleotides long, although their lengths ranged from 1 to 9 bases. The fraction of intact RNA primers 1 to 4 ribonucleotides long was 14 to 73%, depending on the template used. RNA primer length varied among primers initiated at the same nucleotide, as well as with primers initiated at different sites. Thus, the size of an RNA primer depended on template sequence. Initiation sites were identified by mapping 5' ends of nascent RNA-DNA chains on the template sequence, identifying the 5'-terminal ribonucleotide, and partially sequencing one RNA primer. A total of 56 initiation events were identified on simian virus 40 DNA, an average of 1 every 16 bases. Some sites were preferred over others. A consensus sequence for initiation sites consisted of either 3'-dCTTT or 3'-dCCC centered within 7 to 25 pyrimidine-rich residues; the 5' ends of RNA primers were complementary to the dT or dC. High ATP/GTP ratios promoted initiation of RNA primer synthesis at 3'-dCTTT sites, whereas low ATP/GTP ratios promoted initiation at 3'-dCCC sites. Similarly, polydeoxythymidylic acid and polydeoxycytidylic acid were the only effective homopolymer templates. Thus, both template sequence and ribonucleoside triphosphate concentrations determine which initiation sites are used by DNA primase-DNA polymerase alpha. Remarkably, initiation sites selected in vitro were strikingly different from initiation sites selected during simian virus 40 DNA replication in vivo.

Functional analysis of the role of the A + T-rich region and upstream flanking sequences in simian virus 40 DNA replication

1986 ◽  
Vol 6 (12) ◽  
pp. 4570-4577
Author(s):  
R Gerard ◽  
Y Gluzman
Keyword(s):  
Dna Replication ◽  
Point Mutations ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Rich Region ◽  
Viral Dnas ◽  
Origin Function

One boundary of the minimal origin of replication of simian virus 40 DNA lies within the A + T-rich region. Deletion of only a few bases into the adenine-thymine (AT) stretch results in a DNA template which is defective for replication both in vivo and in vitro (B. Stillman, R. D. Gerard, R. A. Guggenheimer, and Y. Gluzman, EMBO J. 4:2933-2939, 1985). In the present study, such deletion mutations have been reconstructed into a simian virus 40 genome containing an intact early promoter-enhancer region. The resulting mutants synthesized wild-type levels of T antigen, but were defective for replication and would not form plaques on CV-1 monkey cells. Replication-competent phenotypic revertants were selected after transfection of large quantities of the replication-defective viral DNAs into CV-1 cells. DNA sequence analysis showed that most of these revertants contained insertions or point mutations which partially regenerate the length of the AT stretch. These genotypic alterations were shown to be responsible for the revertant phenotype by replication analysis in vivo of subcloned revertant origin fragments. In general, our results emphasize the importance of the AT region to simian virus 40 origin function. However, one revertant retained the altered AT region but deleted six nucleotides upstream. Experiments using this mutant indicate that the 21-base-pair repeats identified as part of the early transcriptional promoter may compensate for defects in simian virus 40 DNA replication in vivo caused by mutations in the A + T-rich region when positioned at an appropriate distance from the core origin.

scholarly journals Cloned mouse DNA fragments can replicate in a simian virus 40 T antigen-dependent system in vivo and in vitro.

1985 ◽  
Vol 5 (3) ◽  
pp. 563-568 ◽  
Author(s):  
H Ariga ◽  
Z Tsuchihashi ◽  
M Naruto ◽  
M Yamada
Keyword(s):  
Dna Replication ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Yeast Cells ◽  
Dna Fragments ◽  
Replication System ◽  
Sv40 Dna

Mouse liver DNA was cut out with BamHI and cloned into YIp5, which contained the URA3 gene of Saccharomyces cerevisiae in pBR322. Of the several plasmids isolated, two plasmids, pMU65 and pMU111, could transform S. cerevisiae from the URA- to the URA+ phenotype and could replicate autonomously within the transformant, indicating that mouse DNA fragments present in pMU65 or pMU111 contain autonomously replicating sequences (ARS) for replication in S. cerevisiae. Furthermore, to determine the correlation between ARS function in yeast cells and that in much higher organisms, we tried to challenge these plasmids with the simian virus 40 (SV40) DNA replication system. Of the two plasmids tested, the EcoRI-BglII region of pMU65 could be hybridized with a chemically synthesized 13-nucleotide fragment corresponding to the origin region of SV40 DNA. Both pMU65 (the EcoRI-BglII region cloned in pBR322) and its subclone pMU65EB could replicate semiconservatively, and initiation of DNA replication started from the EcoRI-BglII region when the replicating activity of these plasmids was tested in the in vitro SV40 DNA replication system we have established before. Furthermore, pMU65 and pMU65EB could replicate autonomously within monkey Cos cells which produce SV40 T antigen constitutively. These results show that a 2.5-kilobase fragment of the EcoRI-BglII region in pMU65 contains the ARS needed for replication in the SV40 DNA replication system.

scholarly journals Origin auxiliary sequences can facilitate initiation of simian virus 40 DNA replication in vitro as they do in vivo.

1989 ◽  
Vol 9 (9) ◽  
pp. 3593-3602 ◽  
Author(s):  
Z S Guo ◽  
C Gutierrez ◽  
U Heine ◽  
J M Sogo ◽  
M L Depamphilis
Keyword(s):  
Dna Replication ◽  
Tumor Antigen ◽  
Simian Virus 40 ◽  
Cell Extract ◽  
Simian Virus ◽  
Initiation Factor ◽  
Large Tumor ◽  
Infected Monkey

Initiation of simian virus 40 (SV40) DNA replication is facilitated by two auxiliary sequences that flank the minimally required origin (ori) core sequence. In monkey cells, the replication rate of each of the four ori configurations changed with time after transfection in a characteristic pattern. This pattern was reproduced in an extract from SV40-infected monkey cells by varying the ratio of DNA substrate to cell extract; DNA replication in vitro depended on ori auxiliary sequences to the same extent as they did in vivo. Facilitation by ori auxiliary sequences was lost at high ratios of DNA to cell extract, revealing that the activity of these sequences required either multiple initiation factors or a molar excess of one initiation factor bound to ori. This parameter, together with ionic strength and the method used to measure DNA replication, determined the level of facilitation by ori auxiliary sequences in vitro. The activity of ori auxiliary sequences was not diminished in vivo or in vitro by increasing amounts of large tumor antigen. Therefore, ori auxiliary sequences promoted initiation of replication at some step after tumor antigen binding to ori. Furthermore, although cellular factors could modulate the activity of ori auxiliary sequences in vitro, these factors did not appear to involve nucleosome assembly because no correlation was observed between the number of nucleosomes assembled per DNA molecule and facilitation by ori auxiliary sequences. These results demonstrate that SV40 ori auxiliary sequences can function in vitro as they do in vivo and begin to elucidate their role in initiating DNA replication.

scholarly journals Human Topoisomerase I Promotes Initiation of Simian Virus 40 DNA Replication In Vitro

1999 ◽  
Vol 19 (3) ◽  
pp. 1686-1694 ◽  
Author(s):  
Pamela W. Trowbridge ◽  
Rupa Roy ◽  
Daniel T. Simmons
Keyword(s):  
Dna Replication ◽  
Topoisomerase I ◽  
Competitive Inhibition ◽  
Active Form ◽  
Direct Interaction ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Stimulation Of

ABSTRACT Addition of purified human topoisomerase I (topo I) to simian virus 40 T antigen-driven in vitro DNA replication reactions performed with topo I-deficient extracts results in a greater than 10-fold stimulation of completed molecules as well as a more than 3-fold enhancement of overall DNA replication. To further characterize this stimulation, we first demonstrate that bovine topo I but not Escherichia coli topo I can also enhance DNA replication. By using several human topo I mutants, we show that a catalytically active form of topo I is required. To delineate whether topo I influences the initiation or the elongation step of replication, we performed delayed pulse, pulse-chase, and delayed pulse-chase experiments. The results illustrate that topo I cannot promote the completion of partially replicated molecules but is needed from the beginning of the reaction to initiate replication. Competitive inhibition experiments with the topo I binding T antigen fragment 1-246T and a catalytically inactive topo I mutant suggest that part of topo I’s stimulation of replication is mediated through a direct interaction with T antigen. Collectively, our data indicate that topo I enhances the synthesis of fully replicated DNA molecules by forming essential interactions with T antigen and stimulating initiation.

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