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

Initiation of DNA replication is tightly controlled during the cell cycle to maintain genome integrity. In order to directly study this control we have previously established a cell-free system from human cells that initiates semi-conservative DNA replication. Template nuclei are isolated from cells...

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Detalles Bibliográficos
Autores principales: Marheineke, Kathrin, Hyrien, Olivier, Krude, Torsten
Formato: Texto
Lenguaje:English
Publicado: Oxford University Press 2005
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1310965/
https://www.ncbi.nlm.nih.gov/pubmed/16332696
http://dx.doi.org/10.1093/nar/gki994
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author Marheineke, Kathrin
Hyrien, Olivier
Krude, Torsten
author_facet Marheineke, Kathrin
Hyrien, Olivier
Krude, Torsten
author_sort Marheineke, Kathrin
collection PubMed
description Initiation of DNA replication is tightly controlled during the cell cycle to maintain genome integrity. In order to directly study this control we have previously established a cell-free system from human cells that initiates semi-conservative DNA replication. Template nuclei are isolated from cells synchronized in late G(1) phase by mimosine. We have now used DNA combing to investigate initiation and further progression of DNA replication forks in this human in vitro system at single molecule level. We obtained direct evidence for bidirectional initiation of divergently moving replication forks in vitro. We assessed quantitatively replication fork initiation patterns, fork movement rates and overall fork density. Individual replication forks progress at highly heterogeneous rates (304 ± 162 bp/min) and the two forks emanating from a single origin progress independently from each other. Fork progression rates also change at the single fork level, suggesting that replication fork stalling occurs. DNA combing provides a powerful approach to analyse dynamics of human DNA replication in vitro.
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spelling pubmed-13109652005-12-12 Visualization of bidirectional initiation of chromosomal DNA replication in a human cell free system Marheineke, Kathrin Hyrien, Olivier Krude, Torsten Nucleic Acids Res Article Initiation of DNA replication is tightly controlled during the cell cycle to maintain genome integrity. In order to directly study this control we have previously established a cell-free system from human cells that initiates semi-conservative DNA replication. Template nuclei are isolated from cells synchronized in late G(1) phase by mimosine. We have now used DNA combing to investigate initiation and further progression of DNA replication forks in this human in vitro system at single molecule level. We obtained direct evidence for bidirectional initiation of divergently moving replication forks in vitro. We assessed quantitatively replication fork initiation patterns, fork movement rates and overall fork density. Individual replication forks progress at highly heterogeneous rates (304 ± 162 bp/min) and the two forks emanating from a single origin progress independently from each other. Fork progression rates also change at the single fork level, suggesting that replication fork stalling occurs. DNA combing provides a powerful approach to analyse dynamics of human DNA replication in vitro. Oxford University Press 2005 2005-12-06 /pmc/articles/PMC1310965/ /pubmed/16332696 http://dx.doi.org/10.1093/nar/gki994 Text en © The Author 2005. Published by Oxford University Press. All rights reserved
spellingShingle Article
Marheineke, Kathrin
Hyrien, Olivier
Krude, Torsten
Visualization of bidirectional initiation of chromosomal DNA replication in a human cell free system
title Visualization of bidirectional initiation of chromosomal DNA replication in a human cell free system
title_full Visualization of bidirectional initiation of chromosomal DNA replication in a human cell free system
title_fullStr Visualization of bidirectional initiation of chromosomal DNA replication in a human cell free system
title_full_unstemmed Visualization of bidirectional initiation of chromosomal DNA replication in a human cell free system
title_short Visualization of bidirectional initiation of chromosomal DNA replication in a human cell free system
title_sort visualization of bidirectional initiation of chromosomal dna replication in a human cell free system
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1310965/
https://www.ncbi.nlm.nih.gov/pubmed/16332696
http://dx.doi.org/10.1093/nar/gki994
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AT krudetorsten visualizationofbidirectionalinitiationofchromosomaldnareplicationinahumancellfreesystem