scholarly journals Visualization of bidirectional initiation of chromosomal DNA replication in a human cell free system

2005 ◽  
Vol 33 (21) ◽  
pp. 6931-6941 ◽  
Author(s):  
K. Marheineke
Keyword(s):  
Dna Replication ◽  
Human Cell ◽  
Chromosomal Dna ◽  
Free System ◽  
Cell Free System

scholarly journals Cyclin/Cdk-Dependent Initiation of DNA Replication in a Human Cell-Free System

Cell ◽  
1997 ◽  
Vol 88 (1) ◽  
pp. 109-119 ◽  
Author(s):  
Torsten Krude ◽  
Mark Jackman ◽  
Jonathon Pines ◽  
Ronald A Laskey
Keyword(s):  
Dna Replication ◽  
Human Cell ◽  
Free System ◽  
Cell Free System ◽  
Initiation Of Dna Replication

Efficient long DNA gap-filling in a mammalian cell-free system: A potential new in vitro DNA replication assay

Biochimie ◽  
2013 ◽  
Vol 95 (2) ◽  
pp. 320-328
Author(s):  
Seiki Nakao ◽  
Sufang Zhang ◽  
Markku Vaara ◽  
Juhani E. Syväoja ◽  
Marietta Y. Lee ◽  
...  
Keyword(s):  
Dna Replication ◽  
Mammalian Cell ◽  
Gap Filling ◽  
Free System ◽  
Cell Free System ◽  
Replication Assay ◽  
System A

scholarly journals Functional Requirement of Noncoding Y RNAs for Human Chromosomal DNA Replication

2006 ◽  
Vol 26 (18) ◽  
pp. 6993-7004 ◽  
Author(s):  
Christo P. Christov ◽  
Timothy J. Gardiner ◽  
Dávid Szüts ◽  
Torsten Krude
Keyword(s):  
Dna Replication ◽  
Functional Characterization ◽  
Noncoding Rnas ◽  
Biological Processes ◽  
Replication Forks ◽  
Chromosomal Dna ◽  
Free System ◽  
Specific Degradation

ABSTRACT Noncoding RNAs are recognized increasingly as important regulators of fundamental biological processes, such as gene expression and development, in eukaryotes. We report here the identification and functional characterization of the small noncoding human Y RNAs (hY RNAs) as novel factors for chromosomal DNA replication in a human cell-free system. In addition to protein fractions, hY RNAs are essential for the establishment of active chromosomal DNA replication forks in template nuclei isolated from late-G1-phase human cells. Specific degradation of hY RNAs leads to the inhibition of semiconservative DNA replication in late-G1-phase template nuclei. This inhibition is negated by resupplementation of hY RNAs. All four hY RNAs (hY1, hY3, hY4, and hY5) can functionally substitute for each other in this system. Mutagenesis of hY1 RNA showed that the binding site for Ro60 protein, which is required for Ro RNP assembly, is not essential for DNA replication. Degradation of hY1 RNA in asynchronously proliferating HeLa cells by RNA interference reduced the percentages of cells incorporating bromodeoxyuridine in vivo. These experiments implicate a functional role for hY RNAs in human chromosomal DNA replication.

scholarly journals Simian virus 40 DNA replication in vitro: specificity of initiation and evidence for bidirectional replication.

1985 ◽  
Vol 5 (6) ◽  
pp. 1238-1246 ◽  
Author(s):  
J J Li ◽  
T J Kelly
Keyword(s):  
Dna Replication ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Dna Molecules ◽  
Free System ◽  
Cell Free System ◽  
Cell Extracts ◽  
Sv40 Origin

We recently described a soluble cell-free system derived from monkey cells that is capable of replicating exogenous plasmid DNA molecules containing the simian virus 40 (SV40) origin of replication (J.J. Li, and T.J. Kelly, Proc. Natl. Acad. Sci. U.S.A. 81:6973-6977, 1984). Replication in the system is completely dependent upon the addition of the SV40 large T antigen. In this report we describe additional properties of the in vitro replication reaction. Extracts prepared from cells of several nonsimian species were tested for the ability to support origin-dependent replication in the presence of T antigen. The activities of extracts derived from human cell lines HeLa and 293 were approximately the same as those of monkey cell extracts. Chinese hamster ovary cell extracts also supported SV40 DNA replication in vitro, but the extent of replication was approximately 1% of that observed with human or monkey cell extracts. No replication activity was detectable in extracts derived from BALB/3T3 mouse cells. The ability of these extracts to support replication in vitro closely parallels the ability of the same cells to support replication in vivo. We also examined the ability of various DNA molecules containing sequences homologous to the SV40 origin to serve as templates in the cell-free system. Plasmids containing the origins of human papovaviruses BKV and JCV replicated with an efficiency 10 to 20% of that of plasmids containing the SV40 origin. Plasmids containing Alu repeat sequences (BLUR8) did not support detectable DNA replication in vitro. Circular DNA molecules were found to be the best templates for DNA replication in the cell-free system; however, linear DNA molecules containing the SV40 origin also replicated to a significant extent (10 to 20% of circular molecules). Finally, electron microscopy of replication intermediates demonstrated that the initiation of DNA synthesis in vivo takes place at a unique site corresponding to the in vivo origin and that replication is bidirectional. These findings provide further evidence that replication in the cell-free system faithfully mimics SV40 DNA replication in vivo.

Simian virus 40 DNA replication in vitro: specificity of initiation and evidence for bidirectional replication

1985 ◽  
Vol 5 (6) ◽  
pp. 1238-1246
Author(s):  
J J Li ◽  
T J Kelly
Keyword(s):  
Dna Replication ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Dna Molecules ◽  
Free System ◽  
Cell Free System ◽  
Cell Extracts ◽  
Sv40 Origin

We recently described a soluble cell-free system derived from monkey cells that is capable of replicating exogenous plasmid DNA molecules containing the simian virus 40 (SV40) origin of replication (J.J. Li, and T.J. Kelly, Proc. Natl. Acad. Sci. U.S.A. 81:6973-6977, 1984). Replication in the system is completely dependent upon the addition of the SV40 large T antigen. In this report we describe additional properties of the in vitro replication reaction. Extracts prepared from cells of several nonsimian species were tested for the ability to support origin-dependent replication in the presence of T antigen. The activities of extracts derived from human cell lines HeLa and 293 were approximately the same as those of monkey cell extracts. Chinese hamster ovary cell extracts also supported SV40 DNA replication in vitro, but the extent of replication was approximately 1% of that observed with human or monkey cell extracts. No replication activity was detectable in extracts derived from BALB/3T3 mouse cells. The ability of these extracts to support replication in vitro closely parallels the ability of the same cells to support replication in vivo. We also examined the ability of various DNA molecules containing sequences homologous to the SV40 origin to serve as templates in the cell-free system. Plasmids containing the origins of human papovaviruses BKV and JCV replicated with an efficiency 10 to 20% of that of plasmids containing the SV40 origin. Plasmids containing Alu repeat sequences (BLUR8) did not support detectable DNA replication in vitro. Circular DNA molecules were found to be the best templates for DNA replication in the cell-free system; however, linear DNA molecules containing the SV40 origin also replicated to a significant extent (10 to 20% of circular molecules). Finally, electron microscopy of replication intermediates demonstrated that the initiation of DNA synthesis in vivo takes place at a unique site corresponding to the in vivo origin and that replication is bidirectional. These findings provide further evidence that replication in the cell-free system faithfully mimics SV40 DNA replication in vivo.

Sign in / Sign up

Export Citation Format

Share Document