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Efficient Arrangement of the Replication Fork Trap for In Vitro Propagation of Monomeric Circular DNA in the Chromosome-Replication Cycle Reaction

Propagation of genetic information is a fundamental prerequisite for living cells. We recently developed the replication cycle reaction (RCR), an in vitro reaction for circular DNA propagation, by reconstitution of the replication cycle of the Escherichia coli chromosome. In RCR, two replication for...

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Autores principales: Hasebe, Tomonori, Narita, Kouhei, Hidaka, Shiomi, Su’etsugu, Masayuki
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6315707/
https://www.ncbi.nlm.nih.gov/pubmed/30257439
http://dx.doi.org/10.3390/life8040043
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author Hasebe, Tomonori
Narita, Kouhei
Hidaka, Shiomi
Su’etsugu, Masayuki
author_facet Hasebe, Tomonori
Narita, Kouhei
Hidaka, Shiomi
Su’etsugu, Masayuki
author_sort Hasebe, Tomonori
collection PubMed
description Propagation of genetic information is a fundamental prerequisite for living cells. We recently developed the replication cycle reaction (RCR), an in vitro reaction for circular DNA propagation, by reconstitution of the replication cycle of the Escherichia coli chromosome. In RCR, two replication forks proceed bidirectionally from the replication origin, oriC, and meet at a region opposite oriC, yielding two copies of circular DNA. Although RCR essentially propagates supercoiled monomers, concatemer byproducts are also produced due to inefficient termination of the replication fork progression. Here, we examined the effect of the Tus-ter replication fork trap in RCR. Unexpectedly, when the fork traps were placed opposite oriC, mimicking their arrangement on the chromosome, the propagation of circular DNA was inhibited. On the other hand, fork traps flanking oriC allowed efficient propagation of circular DNA and repressed concatemer production. These findings suggest that collision of the two convergence forks through the fork trap is detrimental to repetition of the replication cycle. We further demonstrate that this detrimental effect was rescued by the UvrD helicase. These results provide insights into the way in which circular DNA monomers replicate repetitively without generating concatemers.
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spelling pubmed-63157072019-01-10 Efficient Arrangement of the Replication Fork Trap for In Vitro Propagation of Monomeric Circular DNA in the Chromosome-Replication Cycle Reaction Hasebe, Tomonori Narita, Kouhei Hidaka, Shiomi Su’etsugu, Masayuki Life (Basel) Article Propagation of genetic information is a fundamental prerequisite for living cells. We recently developed the replication cycle reaction (RCR), an in vitro reaction for circular DNA propagation, by reconstitution of the replication cycle of the Escherichia coli chromosome. In RCR, two replication forks proceed bidirectionally from the replication origin, oriC, and meet at a region opposite oriC, yielding two copies of circular DNA. Although RCR essentially propagates supercoiled monomers, concatemer byproducts are also produced due to inefficient termination of the replication fork progression. Here, we examined the effect of the Tus-ter replication fork trap in RCR. Unexpectedly, when the fork traps were placed opposite oriC, mimicking their arrangement on the chromosome, the propagation of circular DNA was inhibited. On the other hand, fork traps flanking oriC allowed efficient propagation of circular DNA and repressed concatemer production. These findings suggest that collision of the two convergence forks through the fork trap is detrimental to repetition of the replication cycle. We further demonstrate that this detrimental effect was rescued by the UvrD helicase. These results provide insights into the way in which circular DNA monomers replicate repetitively without generating concatemers. MDPI 2018-09-25 /pmc/articles/PMC6315707/ /pubmed/30257439 http://dx.doi.org/10.3390/life8040043 Text en © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Hasebe, Tomonori
Narita, Kouhei
Hidaka, Shiomi
Su’etsugu, Masayuki
Efficient Arrangement of the Replication Fork Trap for In Vitro Propagation of Monomeric Circular DNA in the Chromosome-Replication Cycle Reaction
title Efficient Arrangement of the Replication Fork Trap for In Vitro Propagation of Monomeric Circular DNA in the Chromosome-Replication Cycle Reaction
title_full Efficient Arrangement of the Replication Fork Trap for In Vitro Propagation of Monomeric Circular DNA in the Chromosome-Replication Cycle Reaction
title_fullStr Efficient Arrangement of the Replication Fork Trap for In Vitro Propagation of Monomeric Circular DNA in the Chromosome-Replication Cycle Reaction
title_full_unstemmed Efficient Arrangement of the Replication Fork Trap for In Vitro Propagation of Monomeric Circular DNA in the Chromosome-Replication Cycle Reaction
title_short Efficient Arrangement of the Replication Fork Trap for In Vitro Propagation of Monomeric Circular DNA in the Chromosome-Replication Cycle Reaction
title_sort efficient arrangement of the replication fork trap for in vitro propagation of monomeric circular dna in the chromosome-replication cycle reaction
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6315707/
https://www.ncbi.nlm.nih.gov/pubmed/30257439
http://dx.doi.org/10.3390/life8040043
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