The Consequences of Replicating in the Wrong Orientation: Bacterial Chromosome Duplication without an Active Replication Origin

ABSTRACT Chromosome replication is regulated in all organisms at the assembly stage of the replication machinery at specific origins. In Escherichia coli, the DnaA initiator protein regulates the assembly of replication forks at oriC. This regulation can be undermined by defects in nucleic acid meta...

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Autores principales:	Juachi U. Dimude, Anna Stockum, Sarah L. Midgley-Smith, Amy L. Upton, Helen A. Foster, Arshad Khan, Nigel J. Saunders, Renata Retkute, Christian J. Rudolph
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2015
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/79b4d77668a248bfa16f07df16ae890e
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spelling	oai:doaj.org-article:79b4d77668a248bfa16f07df16ae890e2021-11-15T15:41:24ZThe Consequences of Replicating in the Wrong Orientation: Bacterial Chromosome Duplication without an Active Replication Origin10.1128/mBio.01294-152150-7511https://doaj.org/article/79b4d77668a248bfa16f07df16ae890e2015-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01294-15https://doaj.org/toc/2150-7511ABSTRACT Chromosome replication is regulated in all organisms at the assembly stage of the replication machinery at specific origins. In Escherichia coli, the DnaA initiator protein regulates the assembly of replication forks at oriC. This regulation can be undermined by defects in nucleic acid metabolism. In cells lacking RNase HI, replication initiates independently of DnaA and oriC, presumably at persisting R-loops. A similar mechanism was assumed for origin-independent synthesis in cells lacking RecG. However, recently we suggested that this synthesis initiates at intermediates resulting from replication fork fusions. Here we present data suggesting that in cells lacking RecG or RNase HI, origin-independent synthesis arises by different mechanisms, indicative of these two proteins having different roles in vivo. Our data support the idea that RNase HI processes R-loops, while RecG is required to process replication fork fusion intermediates. However, regardless of how origin-independent synthesis is initiated, a fraction of forks will proceed in an orientation opposite to normal. We show that the resulting head-on encounters with transcription threaten cell viability, especially if taking place in highly transcribed areas. Thus, despite their different functions, RecG and RNase HI are both important factors for maintaining replication control and orientation. Their absence causes severe replication problems, highlighting the advantages of the normal chromosome arrangement, which exploits a single origin to control the number of forks and their orientation relative to transcription, and a defined termination area to contain fork fusions. Any changes to this arrangement endanger cell cycle control, chromosome dynamics, and, ultimately, cell viability. IMPORTANCE Cell division requires unwinding of millions of DNA base pairs to generate the template for RNA transcripts as well as chromosome replication. As both processes use the same template, frequent clashes are unavoidable. To minimize the impact of these clashes, transcription and replication in bacteria follow the same directionality, thereby avoiding head-on collisions. This codirectionality is maintained by a strict regulation of where replication is started. We have used Escherichia coli as a model to investigate cells in which the defined location of replication initiation is compromised. In cells lacking either RNase HI or RecG, replication initiates away from the defined replication origin, and we discuss the different mechanisms by which this synthesis arises. In addition, the resulting forks proceed in a direction opposite to normal, thereby inducing head-on collisions between transcription and replication, and we show that the resulting consequences are severe enough to threaten the viability of cells.Juachi U. DimudeAnna StockumSarah L. Midgley-SmithAmy L. UptonHelen A. FosterArshad KhanNigel J. SaundersRenata RetkuteChristian J. RudolphAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 6, Iss 6 (2015)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Juachi U. Dimude Anna Stockum Sarah L. Midgley-Smith Amy L. Upton Helen A. Foster Arshad Khan Nigel J. Saunders Renata Retkute Christian J. Rudolph The Consequences of Replicating in the Wrong Orientation: Bacterial Chromosome Duplication without an Active Replication Origin
description	ABSTRACT Chromosome replication is regulated in all organisms at the assembly stage of the replication machinery at specific origins. In Escherichia coli, the DnaA initiator protein regulates the assembly of replication forks at oriC. This regulation can be undermined by defects in nucleic acid metabolism. In cells lacking RNase HI, replication initiates independently of DnaA and oriC, presumably at persisting R-loops. A similar mechanism was assumed for origin-independent synthesis in cells lacking RecG. However, recently we suggested that this synthesis initiates at intermediates resulting from replication fork fusions. Here we present data suggesting that in cells lacking RecG or RNase HI, origin-independent synthesis arises by different mechanisms, indicative of these two proteins having different roles in vivo. Our data support the idea that RNase HI processes R-loops, while RecG is required to process replication fork fusion intermediates. However, regardless of how origin-independent synthesis is initiated, a fraction of forks will proceed in an orientation opposite to normal. We show that the resulting head-on encounters with transcription threaten cell viability, especially if taking place in highly transcribed areas. Thus, despite their different functions, RecG and RNase HI are both important factors for maintaining replication control and orientation. Their absence causes severe replication problems, highlighting the advantages of the normal chromosome arrangement, which exploits a single origin to control the number of forks and their orientation relative to transcription, and a defined termination area to contain fork fusions. Any changes to this arrangement endanger cell cycle control, chromosome dynamics, and, ultimately, cell viability. IMPORTANCE Cell division requires unwinding of millions of DNA base pairs to generate the template for RNA transcripts as well as chromosome replication. As both processes use the same template, frequent clashes are unavoidable. To minimize the impact of these clashes, transcription and replication in bacteria follow the same directionality, thereby avoiding head-on collisions. This codirectionality is maintained by a strict regulation of where replication is started. We have used Escherichia coli as a model to investigate cells in which the defined location of replication initiation is compromised. In cells lacking either RNase HI or RecG, replication initiates away from the defined replication origin, and we discuss the different mechanisms by which this synthesis arises. In addition, the resulting forks proceed in a direction opposite to normal, thereby inducing head-on collisions between transcription and replication, and we show that the resulting consequences are severe enough to threaten the viability of cells.
format	article
author	Juachi U. Dimude Anna Stockum Sarah L. Midgley-Smith Amy L. Upton Helen A. Foster Arshad Khan Nigel J. Saunders Renata Retkute Christian J. Rudolph
author_facet	Juachi U. Dimude Anna Stockum Sarah L. Midgley-Smith Amy L. Upton Helen A. Foster Arshad Khan Nigel J. Saunders Renata Retkute Christian J. Rudolph
author_sort	Juachi U. Dimude
title	The Consequences of Replicating in the Wrong Orientation: Bacterial Chromosome Duplication without an Active Replication Origin
title_short	The Consequences of Replicating in the Wrong Orientation: Bacterial Chromosome Duplication without an Active Replication Origin
title_full	The Consequences of Replicating in the Wrong Orientation: Bacterial Chromosome Duplication without an Active Replication Origin
title_fullStr	The Consequences of Replicating in the Wrong Orientation: Bacterial Chromosome Duplication without an Active Replication Origin
title_full_unstemmed	The Consequences of Replicating in the Wrong Orientation: Bacterial Chromosome Duplication without an Active Replication Origin
title_sort	consequences of replicating in the wrong orientation: bacterial chromosome duplication without an active replication origin
publisher	American Society for Microbiology
publishDate	2015
url	https://doaj.org/article/79b4d77668a248bfa16f07df16ae890e
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The Consequences of Replicating in the Wrong Orientation: Bacterial Chromosome Duplication without an Active Replication Origin

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