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Arrest of Viral Proliferation by Ectopic Copies of Its Cognate Replication Origin.

Valenzuela MS, Sharan C - Genes (Basel) (2015)

Bottom Line: Bacteriophage λ was used as a model system.By measuring the effect on the host growth, viral production, and electro-microscopic visualization of the resulting λ replicative intermediates, we concluded that the ectopic copies had prevented the normal initiation step of λ DNA replication.These results suggest that DNA decoys encoding viral origins could constitute effective tools to specifically arrest viral proliferation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cancer Biology, School of Medicine, Meharry Medical College, 1005 D.B. Todd Jr. Boulevard, Nashville, TN 37208, USA. mvalenzuela@mmc.edu.

ABSTRACT
The initiation step of DNA replication is the crucial determinant of proliferation in all organisms. This step depends on the specific interaction of DNA sequences present at origins of DNA replication and their cognate activators. We wished to explore the hypothesis that the presence of ectopic origin copies may interfere with proper genome duplication. Bacteriophage λ was used as a model system. To this end, the outcome of an infection of an E. coli strain harboring ectopic copies of the λ origin region was analyzed. By measuring the effect on the host growth, viral production, and electro-microscopic visualization of the resulting λ replicative intermediates, we concluded that the ectopic copies had prevented the normal initiation step of λ DNA replication. These results suggest that DNA decoys encoding viral origins could constitute effective tools to specifically arrest viral proliferation.

No MeSH data available.


Related in: MedlinePlus

DNA fractionation after a second CsCl equilibrium density centrifugation of intracellular 3H-labeled λ DNA isolated from infected HBT or HBT(pOri1). CsCl fractions containing viral DNA, and separated from bacterial DNA through a first CsCl gradient, were pooled and run through a second CsCl in order to isolate newly synthesized DNA. At the end of the run, DNA was fractionated and the radioactivity in each fraction, from the lowest density fraction (fraction #1) to the highest density (fraction #30) was determined by scintillation counting. Under the conditions of centrifugation the density of LL DNA is around fractions 15–16. Note that the DNA shoulder past fraction 16 is more prominent in HBT compared to HBT(pOri1).
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genes-06-00436-f007: DNA fractionation after a second CsCl equilibrium density centrifugation of intracellular 3H-labeled λ DNA isolated from infected HBT or HBT(pOri1). CsCl fractions containing viral DNA, and separated from bacterial DNA through a first CsCl gradient, were pooled and run through a second CsCl in order to isolate newly synthesized DNA. At the end of the run, DNA was fractionated and the radioactivity in each fraction, from the lowest density fraction (fraction #1) to the highest density (fraction #30) was determined by scintillation counting. Under the conditions of centrifugation the density of LL DNA is around fractions 15–16. Note that the DNA shoulder past fraction 16 is more prominent in HBT compared to HBT(pOri1).

Mentions: When λ DNA was isolated from infected HBT or HBT(pOri1) and purified through a second CsCl gradient, it was apparent that the intracellular λ DNA obtained after HBT infection was slightly more skewed toward heavier CsCl densities compared to the one obtained from the infected HBT(pOri1) strain (Figure 7). This suggested that the extent of λ DNA replication in HBT had been comparatively greater than in HBT(pOri1). When selected fractions (between DNA densities corresponding to LL and HL) from these two preparations were analyzed by electron microscopy, about 60% of all λ DNA structures observed (n = 138) in infected HBT represented normal theta replicative intermediates [9,10], whereas in HBT(pOri1) only 8% of all structures (n = 107) belonged to this category. These results confirmed our prediction that in HBT(pOri1) λ DNA replication had been impaired. More importantly, in HBT(pOri1) about 66% of all the λ DNA molecules observed contained a “D-loop” like structure (a representative molecule is shown in Figure 8). In contrast, no such structures were observed in preparations obtained from infected HBT. D-loops had been typically observed in λ mutants (such as O and P gene mutants) that were defective in the initiation step of DNA replication [22]. Treatment of these structures with RNaseH caused the disappearance of these D-loops indicating that one of the strands in the loop was made up of RNA [8]. These structures, also found under replicative stress conditions [27] have been interpreted as DNA molecules attempting to use transcription to initiate DNA replication, Supporting this view is the finding that the location of the D-loops in λO− mutants occurs around highly transcribed regions [22]. To ascertain that the “D-loop” structures we had observed after λ infection of HBT(pOri1) had a similar composition we subjected our samples to a similar RNAseH treatment. Electron microscopic observation of the resulting DNA samples yielded no loop structures (n = 137) demonstrating that DNA structures we found also contained RNA at the “D-loops” thus furthering the similarity between the outcome of λ infection of HBT(pOri1) and λO− infection of E. coli.


Arrest of Viral Proliferation by Ectopic Copies of Its Cognate Replication Origin.

Valenzuela MS, Sharan C - Genes (Basel) (2015)

DNA fractionation after a second CsCl equilibrium density centrifugation of intracellular 3H-labeled λ DNA isolated from infected HBT or HBT(pOri1). CsCl fractions containing viral DNA, and separated from bacterial DNA through a first CsCl gradient, were pooled and run through a second CsCl in order to isolate newly synthesized DNA. At the end of the run, DNA was fractionated and the radioactivity in each fraction, from the lowest density fraction (fraction #1) to the highest density (fraction #30) was determined by scintillation counting. Under the conditions of centrifugation the density of LL DNA is around fractions 15–16. Note that the DNA shoulder past fraction 16 is more prominent in HBT compared to HBT(pOri1).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4488673&req=5

genes-06-00436-f007: DNA fractionation after a second CsCl equilibrium density centrifugation of intracellular 3H-labeled λ DNA isolated from infected HBT or HBT(pOri1). CsCl fractions containing viral DNA, and separated from bacterial DNA through a first CsCl gradient, were pooled and run through a second CsCl in order to isolate newly synthesized DNA. At the end of the run, DNA was fractionated and the radioactivity in each fraction, from the lowest density fraction (fraction #1) to the highest density (fraction #30) was determined by scintillation counting. Under the conditions of centrifugation the density of LL DNA is around fractions 15–16. Note that the DNA shoulder past fraction 16 is more prominent in HBT compared to HBT(pOri1).
Mentions: When λ DNA was isolated from infected HBT or HBT(pOri1) and purified through a second CsCl gradient, it was apparent that the intracellular λ DNA obtained after HBT infection was slightly more skewed toward heavier CsCl densities compared to the one obtained from the infected HBT(pOri1) strain (Figure 7). This suggested that the extent of λ DNA replication in HBT had been comparatively greater than in HBT(pOri1). When selected fractions (between DNA densities corresponding to LL and HL) from these two preparations were analyzed by electron microscopy, about 60% of all λ DNA structures observed (n = 138) in infected HBT represented normal theta replicative intermediates [9,10], whereas in HBT(pOri1) only 8% of all structures (n = 107) belonged to this category. These results confirmed our prediction that in HBT(pOri1) λ DNA replication had been impaired. More importantly, in HBT(pOri1) about 66% of all the λ DNA molecules observed contained a “D-loop” like structure (a representative molecule is shown in Figure 8). In contrast, no such structures were observed in preparations obtained from infected HBT. D-loops had been typically observed in λ mutants (such as O and P gene mutants) that were defective in the initiation step of DNA replication [22]. Treatment of these structures with RNaseH caused the disappearance of these D-loops indicating that one of the strands in the loop was made up of RNA [8]. These structures, also found under replicative stress conditions [27] have been interpreted as DNA molecules attempting to use transcription to initiate DNA replication, Supporting this view is the finding that the location of the D-loops in λO− mutants occurs around highly transcribed regions [22]. To ascertain that the “D-loop” structures we had observed after λ infection of HBT(pOri1) had a similar composition we subjected our samples to a similar RNAseH treatment. Electron microscopic observation of the resulting DNA samples yielded no loop structures (n = 137) demonstrating that DNA structures we found also contained RNA at the “D-loops” thus furthering the similarity between the outcome of λ infection of HBT(pOri1) and λO− infection of E. coli.

Bottom Line: Bacteriophage λ was used as a model system.By measuring the effect on the host growth, viral production, and electro-microscopic visualization of the resulting λ replicative intermediates, we concluded that the ectopic copies had prevented the normal initiation step of λ DNA replication.These results suggest that DNA decoys encoding viral origins could constitute effective tools to specifically arrest viral proliferation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cancer Biology, School of Medicine, Meharry Medical College, 1005 D.B. Todd Jr. Boulevard, Nashville, TN 37208, USA. mvalenzuela@mmc.edu.

ABSTRACT
The initiation step of DNA replication is the crucial determinant of proliferation in all organisms. This step depends on the specific interaction of DNA sequences present at origins of DNA replication and their cognate activators. We wished to explore the hypothesis that the presence of ectopic origin copies may interfere with proper genome duplication. Bacteriophage λ was used as a model system. To this end, the outcome of an infection of an E. coli strain harboring ectopic copies of the λ origin region was analyzed. By measuring the effect on the host growth, viral production, and electro-microscopic visualization of the resulting λ replicative intermediates, we concluded that the ectopic copies had prevented the normal initiation step of λ DNA replication. These results suggest that DNA decoys encoding viral origins could constitute effective tools to specifically arrest viral proliferation.

No MeSH data available.


Related in: MedlinePlus