scholarly journals Development of an in vitro bacteriophage N4 DNA replication system.

1986 ◽  
Vol 261 (23) ◽  
pp. 10506-10510
Author(s):  
J K Rist ◽  
M Pearle ◽  
A Sugino ◽  
L B Rothman-Denes
Keyword(s):  
Dna Replication ◽  
Replication System ◽  
Bacteriophage N4

Homologous recombination of monkey alpha-satellite repeats in an in vitro simian virus 40 replication system: possible association of recombination with DNA replication

1994 ◽  
Vol 14 (6) ◽  
pp. 4173-4182
Author(s):  
I Kawasaki ◽  
Y S Bae ◽  
T Eki ◽  
Y Kim ◽  
H Ikeda
Keyword(s):  
Dna Replication ◽  
Homologous Recombination ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Alpha Satellite ◽  
E Coli ◽  
Replication System ◽  
Satellite Repeats

To study homologous recombination between repeated sequences in an in vitro simian virus 40 (SV40) replication system, we constructed a series of substrate DNAs that contain two identical fragments of monkey alpha-satellite repeats. Together with the SV40-pBR322 composite vector encoding Apr and Kmr, the DNAs also contain the Escherichia coli galactokinase gene (galK) positioned between two alpha-satellite fragments. The alpha-satellite sequence used consists of multiple units of tandem 172-bp sequences which differ by microheterogeneity. The substrate DNAs were incubated in an in vitro SV40 DNA replication system and used to transform the E. coli galK strain DH10B after digestion with DpnI. The number of E. coli galK Apr Kmr colonies which contain recombinant DNAs were determined, and their structures were analyzed. Products of equal and unequal crossovers between identical 172-bp sequences and between similar but not identical (homeologous) 172-bp sequences, respectively, were detected, although those of the equal crossover were predominant among all of the galK mutant recombinants. Similar products were also observed in the in vivo experiments with COS1 cells. The in vitro experiments showed that these recombinations were dependent on the presence of both the SV40 origin of DNA replication and SV40 large T antigen. Most of the recombinant DNAs were generated from newly synthesized DpnI-resistant DNAs. These results suggest that the homologous recombination observed in this SV40 system is associated with DNA replication and is suppressed by mismatches in heteroduplexes formed between similar but not identical sequences.

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.

Fidelity of DNA synthesis in a mammalian in vitro replication system

1988 ◽  
Vol 8 (8) ◽  
pp. 3267-3271
Author(s):  
J Hauser ◽  
A S Levine ◽  
K Dixon
Keyword(s):  
Dna Replication ◽  
Shuttle Vector ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Base Substitution ◽  
Replication System ◽  
In Vitro Replication ◽  
Mutation Assay

We have used the simian virus 40 (SV40)-based shuttle vector pZ189 in a forward-mutation assay to determine the fidelity of DNA replication in the in vitro DNA replication system developed by J.J. Li and T.J. Kelly (Proc. Natl. Acad. Sci. USA 81:6973-6977, 1984). We find that very few base substitution errors (approximately 1/180,000 bases incorporated) are made during in vitro replication of the pZ189 vector in a system derived from CV-1 monkey cells. This replication is completely dependent on added SV40 T antigen and presumably reflects synthesis that is initiated at the SV40 replication origin. The observed level of fidelity is far greater than that reported for in vitro replication of DNA by conventionally purified eucaryotic DNA polymerases alpha and beta. Thus, there must be additional cellular factors in the crude in vitro system that serve to enhance the fidelity of DNA replication.

scholarly journals Characterization of Factors that Suppress Linear DNA Replication in SV40 in vitro Replication System.

1993 ◽  
Vol 18 (1) ◽  
pp. 19-32 ◽  
Author(s):  
Shinji Katsura ◽  
Toshihiko Eki ◽  
Hajime Nishimura ◽  
Yasufumi Murakami
Keyword(s):  
Dna Replication ◽  
Replication System ◽  
Linear Dna ◽  
In Vitro Replication

scholarly journals Purification of Host Cell Enzymes Involved in Adeno-Associated Virus DNA Replication

2007 ◽  
Vol 81 (11) ◽  
pp. 5777-5787 ◽  
Author(s):  
Kevin Nash ◽  
Weijun Chen ◽  
William F. McDonald ◽  
Xiaohuai Zhou ◽  
Nicholas Muzyczka
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Dna Helicase ◽  
Strand Displacement ◽  
Adeno Associated Virus ◽  
Replication System ◽  
Factor C

ABSTRACT Adeno-associated virus (AAV) replicates its DNA by a modified rolling-circle mechanism that exclusively uses leading strand displacement synthesis. To identify the enzymes directly involved in AAV DNA replication, we fractionated adenovirus-infected crude extracts and tested them in an in vitro replication system that required the presence of the AAV-encoded Rep protein and the AAV origins of DNA replication, thus faithfully reproducing in vivo viral DNA replication. Fractions that contained replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) were found to be essential for reconstituting AAV DNA replication. These could be replaced by purified PCNA and RFC to retain full activity. We also found that fractions containing polymerase δ, but not polymerase ε or α, were capable of replicating AAV DNA in vitro. This was confirmed when highly purified polymerase δ complex purified from baculovirus expression clones was used. Curiously, as the components of the DNA replication system were purified, neither the cellular single-stranded DNA binding protein (RPA) nor the adenovirus-encoded DNA binding protein was found to be essential for DNA replication; both only modestly stimulated DNA synthesis on an AAV template. Also, in addition to polymerase δ, RFC, and PCNA, an as yet unidentified factor(s) is required for AAV DNA replication, which appeared to be enriched in adenovirus-infected cells. Finally, the absence of any apparent cellular DNA helicase requirement led us to develop an artificial AAV replication system in which polymerase δ, RFC, and PCNA were replaced with T4 DNA polymerase and gp32 protein. This system was capable of supporting AAV DNA replication, demonstrating that under some conditions the Rep helicase activity can function to unwind duplex DNA during strand displacement synthesis.

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