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

Dimude JU, Stockum A, Midgley-Smith SL, Upton AL, Foster HA, Khan A, Saunders NJ, Retkute R, Rudolph CJ - MBio (2015)

Bottom Line: We show that the resulting head-on encounters with transcription threaten cell viability, especially if taking place in highly transcribed areas.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Biosciences, College of Health and Life Sciences, Brunel University London, Uxbridge, United Kingdom.

No MeSH data available.


Related in: MedlinePlus

Effect of chromosome linearization on origin-independent synthesis in recG and rnhA cells. (A and B) Marker frequency analysis of the termination area of E. coli cells in the exponential phase. The number of reads (normalized against the reads for a stationary-phase wild-type control) is plotted against the chromosomal location. Positions of ter sites in the area as well as the integration site of the tos linearization sequence are highlighted. In panels Bi and Ci, data sets for recG and recG tos as well as rnhA and rnhA tos are plotted together for direct comparison. Sequencing templates were isolated from AM1975 (rnhA), RCe605 (rnhA tos), and RCe608 (rnhA tos N15 lysogen). Data for recG (N8226), recG tos (RCe391), and recG tos N15 lysogen (RCe399) were replotted from reference 10. (C) Spot dilution assays to evaluate DnaA-independent growth in dnaA rnhA cells with a linearized chromosome. The strains used were RCe309 (dnaA46 rnhA tus rpo*), JD1160 (dnaA46 rnhA tus rpo* tos), JD1168 (dnaA46 rnhA tus rpo* N15 lysogen), JD1169 (dnaA46 rnhA tus rpo* tos N15 lysogen), RCe268 (dnaA46 recG tus rpo*), RCe385 (dnaA46 recG tus rpo* tos), RCe384 (dnaA46 recG tus rpo* N15 lysogen), and RCe387 (dnaA46 recG tus rpo* tos N15 lysogen).
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fig3: Effect of chromosome linearization on origin-independent synthesis in recG and rnhA cells. (A and B) Marker frequency analysis of the termination area of E. coli cells in the exponential phase. The number of reads (normalized against the reads for a stationary-phase wild-type control) is plotted against the chromosomal location. Positions of ter sites in the area as well as the integration site of the tos linearization sequence are highlighted. In panels Bi and Ci, data sets for recG and recG tos as well as rnhA and rnhA tos are plotted together for direct comparison. Sequencing templates were isolated from AM1975 (rnhA), RCe605 (rnhA tos), and RCe608 (rnhA tos N15 lysogen). Data for recG (N8226), recG tos (RCe391), and recG tos N15 lysogen (RCe399) were replotted from reference 10. (C) Spot dilution assays to evaluate DnaA-independent growth in dnaA rnhA cells with a linearized chromosome. The strains used were RCe309 (dnaA46 rnhA tus rpo*), JD1160 (dnaA46 rnhA tus rpo* tos), JD1168 (dnaA46 rnhA tus rpo* N15 lysogen), JD1169 (dnaA46 rnhA tus rpo* tos N15 lysogen), RCe268 (dnaA46 recG tus rpo*), RCe385 (dnaA46 recG tus rpo* tos), RCe384 (dnaA46 recG tus rpo* N15 lysogen), and RCe387 (dnaA46 recG tus rpo* tos N15 lysogen).

Mentions: In addition, we found that chromosome linearization, which prevents the collision of replisomes, has different effects in cells lacking RecG or RNase HI. Linearization is achieved by introducing the tos linearization sequence from bacteriophage N15 near the chromosome dimer resolution site dif into the chromosome. Subsequent lysogenic infection with N15 results in the expression of the telomerase TelN, which will process the tos linearization sequence (31). We found that insertion of the tos site in rnhA cells resulted in a mild reduction of the overreplication in the termination area, a result not observed in recG cells (cf. Fig. 3Ai and 3Bi). Thus, it appears that integration of the tos linearization cassette interferes with initiation of overreplication in the termination area in rnhA but not recG cells. Linearization of the chromosome, which is clearly visible as a discontinuity in the replication profile (Fig. 3A and B; see Fig. S1 in the supplemental material) (19), caused a reduction of overreplication, both in cells lacking RecG and those lacking RNase HI. However, the resulting profiles look different. Upon linearization, the profile of rnhA cells becomes asymmetric, with no overreplication observed between tos and terA, while some overreplication is observed between terC/B and tos, which appears abruptly cut off at the linearization site (Fig. 3Aii). In contrast, recG cells with a linearized chromosome show elevated marker frequency levels on both sides of the linearization site (Fig. 3Bii).


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

Dimude JU, Stockum A, Midgley-Smith SL, Upton AL, Foster HA, Khan A, Saunders NJ, Retkute R, Rudolph CJ - MBio (2015)

Effect of chromosome linearization on origin-independent synthesis in recG and rnhA cells. (A and B) Marker frequency analysis of the termination area of E. coli cells in the exponential phase. The number of reads (normalized against the reads for a stationary-phase wild-type control) is plotted against the chromosomal location. Positions of ter sites in the area as well as the integration site of the tos linearization sequence are highlighted. In panels Bi and Ci, data sets for recG and recG tos as well as rnhA and rnhA tos are plotted together for direct comparison. Sequencing templates were isolated from AM1975 (rnhA), RCe605 (rnhA tos), and RCe608 (rnhA tos N15 lysogen). Data for recG (N8226), recG tos (RCe391), and recG tos N15 lysogen (RCe399) were replotted from reference 10. (C) Spot dilution assays to evaluate DnaA-independent growth in dnaA rnhA cells with a linearized chromosome. The strains used were RCe309 (dnaA46 rnhA tus rpo*), JD1160 (dnaA46 rnhA tus rpo* tos), JD1168 (dnaA46 rnhA tus rpo* N15 lysogen), JD1169 (dnaA46 rnhA tus rpo* tos N15 lysogen), RCe268 (dnaA46 recG tus rpo*), RCe385 (dnaA46 recG tus rpo* tos), RCe384 (dnaA46 recG tus rpo* N15 lysogen), and RCe387 (dnaA46 recG tus rpo* tos N15 lysogen).
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fig3: Effect of chromosome linearization on origin-independent synthesis in recG and rnhA cells. (A and B) Marker frequency analysis of the termination area of E. coli cells in the exponential phase. The number of reads (normalized against the reads for a stationary-phase wild-type control) is plotted against the chromosomal location. Positions of ter sites in the area as well as the integration site of the tos linearization sequence are highlighted. In panels Bi and Ci, data sets for recG and recG tos as well as rnhA and rnhA tos are plotted together for direct comparison. Sequencing templates were isolated from AM1975 (rnhA), RCe605 (rnhA tos), and RCe608 (rnhA tos N15 lysogen). Data for recG (N8226), recG tos (RCe391), and recG tos N15 lysogen (RCe399) were replotted from reference 10. (C) Spot dilution assays to evaluate DnaA-independent growth in dnaA rnhA cells with a linearized chromosome. The strains used were RCe309 (dnaA46 rnhA tus rpo*), JD1160 (dnaA46 rnhA tus rpo* tos), JD1168 (dnaA46 rnhA tus rpo* N15 lysogen), JD1169 (dnaA46 rnhA tus rpo* tos N15 lysogen), RCe268 (dnaA46 recG tus rpo*), RCe385 (dnaA46 recG tus rpo* tos), RCe384 (dnaA46 recG tus rpo* N15 lysogen), and RCe387 (dnaA46 recG tus rpo* tos N15 lysogen).
Mentions: In addition, we found that chromosome linearization, which prevents the collision of replisomes, has different effects in cells lacking RecG or RNase HI. Linearization is achieved by introducing the tos linearization sequence from bacteriophage N15 near the chromosome dimer resolution site dif into the chromosome. Subsequent lysogenic infection with N15 results in the expression of the telomerase TelN, which will process the tos linearization sequence (31). We found that insertion of the tos site in rnhA cells resulted in a mild reduction of the overreplication in the termination area, a result not observed in recG cells (cf. Fig. 3Ai and 3Bi). Thus, it appears that integration of the tos linearization cassette interferes with initiation of overreplication in the termination area in rnhA but not recG cells. Linearization of the chromosome, which is clearly visible as a discontinuity in the replication profile (Fig. 3A and B; see Fig. S1 in the supplemental material) (19), caused a reduction of overreplication, both in cells lacking RecG and those lacking RNase HI. However, the resulting profiles look different. Upon linearization, the profile of rnhA cells becomes asymmetric, with no overreplication observed between tos and terA, while some overreplication is observed between terC/B and tos, which appears abruptly cut off at the linearization site (Fig. 3Aii). In contrast, recG cells with a linearized chromosome show elevated marker frequency levels on both sides of the linearization site (Fig. 3Bii).

Bottom Line: We show that the resulting head-on encounters with transcription threaten cell viability, especially if taking place in highly transcribed areas.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Biosciences, College of Health and Life Sciences, Brunel University London, Uxbridge, United Kingdom.

No MeSH data available.


Related in: MedlinePlus