Limits...
Two mechanisms coordinate replication termination by the Escherichia coli Tus-Ter complex.

Pandey M, Elshenawy MM, Jergic S, Takahashi M, Dixon NE, Hamdan SM, Patel SS - Nucleic Acids Res. (2015)

Bottom Line: An unpaired C(6) that forms a lock by binding into the cytosine binding pocket of Tus was most effective in arresting the replisome and mutation of C(6) removed the barrier.The polymerase tracking along the template strand traps the C(6) to prevent lock formation; the helicase tracking along the other strand traps the complementary G(6) to aid lock formation.Our results are consistent with the model where strand separation by the helicase unpairs the GC(6) base pair and triggers lock formation immediately before the polymerase can sequester the C(6) base.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Rutgers, the State University of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA pandeyma@rwjms.rutgers.edu.

Show MeSH

Related in: MedlinePlus

Tus–TerB arrest of DNA synthesis by T7 helicase–DNA polymerase at single-nucleotide resolution. (A) Schematic of the experimental design to study replication arrest of T7 helicase-polymerase by Tus–TerB at single-nucleotide resolution using the chemical quenched flow assay. (B) The TerB sequence and the C(6) base. The TerB sequence numbering is followed throughout. (C) High resolution DNA sequencing gel shows progressive strand displacement DNA synthesis by the T7 helicase–polymerase on a fork DNA containing TerB in the non-permissive orientation. Arrows indicate the first arrest position band corresponding to the arrow on the TerB sequence in (B). These reactions were carried out in the quenched flow apparatus (QF) in the presence or in the absence of Tus protein at 150 mM KCl at 0.1 mM dVTPs and 1 mM dTTP. (D) Sequencing gel shows the QF reactions in the presence of Tus at 50 and 300 mM KCl, with all dNTPs at 1 mM. Each time point shown here is an independent reaction. Another QF experiment is also shown in Supplementary Figure S3 and extended time scale experiments are shown in Supplementary Figures S4, S6 and S7.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4499146&req=5

Figure 3: Tus–TerB arrest of DNA synthesis by T7 helicase–DNA polymerase at single-nucleotide resolution. (A) Schematic of the experimental design to study replication arrest of T7 helicase-polymerase by Tus–TerB at single-nucleotide resolution using the chemical quenched flow assay. (B) The TerB sequence and the C(6) base. The TerB sequence numbering is followed throughout. (C) High resolution DNA sequencing gel shows progressive strand displacement DNA synthesis by the T7 helicase–polymerase on a fork DNA containing TerB in the non-permissive orientation. Arrows indicate the first arrest position band corresponding to the arrow on the TerB sequence in (B). These reactions were carried out in the quenched flow apparatus (QF) in the presence or in the absence of Tus protein at 150 mM KCl at 0.1 mM dVTPs and 1 mM dTTP. (D) Sequencing gel shows the QF reactions in the presence of Tus at 50 and 300 mM KCl, with all dNTPs at 1 mM. Each time point shown here is an independent reaction. Another QF experiment is also shown in Supplementary Figure S3 and extended time scale experiments are shown in Supplementary Figures S4, S6 and S7.

Mentions: We next used a rapid quenched flow assay to follow progressive DNA synthesis at single base resolution, allowing us to study the encounter of the T7 helicase–polymerase with Tus–TerB at high spatial and temporal resolution. The 22-bp TerB sequence was introduced in either the permissive or non-permissive orientation at a specific position in the middle of a 60-bp dsDNA region of the synthetic replication fork substrate (Figure 2 and Supplementary Table S1). The replication fork contained a 35-nt 5′-tail for loading of the helicase and a 24-bp primer/template for loading the polymerase (Figure 3A, B). The replication fork was pre-incubated with Tus (380 nM) and the T7 DNA polymerase and helicase in the presence of dTTP, but without Mg2+. These conditions promote preassembly of the replication proteins on the DNA and allow synchronization of the DNA unwinding/synthesis reactions initiated with Mg2+ and dNTPs. The components were mixed in a chemical quenched-flow instrument and quenched after 0.004 to 600 s before analysis of primer extension at single base resolution on a DNA sequencing gel (Figure 3C).


Two mechanisms coordinate replication termination by the Escherichia coli Tus-Ter complex.

Pandey M, Elshenawy MM, Jergic S, Takahashi M, Dixon NE, Hamdan SM, Patel SS - Nucleic Acids Res. (2015)

Tus–TerB arrest of DNA synthesis by T7 helicase–DNA polymerase at single-nucleotide resolution. (A) Schematic of the experimental design to study replication arrest of T7 helicase-polymerase by Tus–TerB at single-nucleotide resolution using the chemical quenched flow assay. (B) The TerB sequence and the C(6) base. The TerB sequence numbering is followed throughout. (C) High resolution DNA sequencing gel shows progressive strand displacement DNA synthesis by the T7 helicase–polymerase on a fork DNA containing TerB in the non-permissive orientation. Arrows indicate the first arrest position band corresponding to the arrow on the TerB sequence in (B). These reactions were carried out in the quenched flow apparatus (QF) in the presence or in the absence of Tus protein at 150 mM KCl at 0.1 mM dVTPs and 1 mM dTTP. (D) Sequencing gel shows the QF reactions in the presence of Tus at 50 and 300 mM KCl, with all dNTPs at 1 mM. Each time point shown here is an independent reaction. Another QF experiment is also shown in Supplementary Figure S3 and extended time scale experiments are shown in Supplementary Figures S4, S6 and S7.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4499146&req=5

Figure 3: Tus–TerB arrest of DNA synthesis by T7 helicase–DNA polymerase at single-nucleotide resolution. (A) Schematic of the experimental design to study replication arrest of T7 helicase-polymerase by Tus–TerB at single-nucleotide resolution using the chemical quenched flow assay. (B) The TerB sequence and the C(6) base. The TerB sequence numbering is followed throughout. (C) High resolution DNA sequencing gel shows progressive strand displacement DNA synthesis by the T7 helicase–polymerase on a fork DNA containing TerB in the non-permissive orientation. Arrows indicate the first arrest position band corresponding to the arrow on the TerB sequence in (B). These reactions were carried out in the quenched flow apparatus (QF) in the presence or in the absence of Tus protein at 150 mM KCl at 0.1 mM dVTPs and 1 mM dTTP. (D) Sequencing gel shows the QF reactions in the presence of Tus at 50 and 300 mM KCl, with all dNTPs at 1 mM. Each time point shown here is an independent reaction. Another QF experiment is also shown in Supplementary Figure S3 and extended time scale experiments are shown in Supplementary Figures S4, S6 and S7.
Mentions: We next used a rapid quenched flow assay to follow progressive DNA synthesis at single base resolution, allowing us to study the encounter of the T7 helicase–polymerase with Tus–TerB at high spatial and temporal resolution. The 22-bp TerB sequence was introduced in either the permissive or non-permissive orientation at a specific position in the middle of a 60-bp dsDNA region of the synthetic replication fork substrate (Figure 2 and Supplementary Table S1). The replication fork contained a 35-nt 5′-tail for loading of the helicase and a 24-bp primer/template for loading the polymerase (Figure 3A, B). The replication fork was pre-incubated with Tus (380 nM) and the T7 DNA polymerase and helicase in the presence of dTTP, but without Mg2+. These conditions promote preassembly of the replication proteins on the DNA and allow synchronization of the DNA unwinding/synthesis reactions initiated with Mg2+ and dNTPs. The components were mixed in a chemical quenched-flow instrument and quenched after 0.004 to 600 s before analysis of primer extension at single base resolution on a DNA sequencing gel (Figure 3C).

Bottom Line: An unpaired C(6) that forms a lock by binding into the cytosine binding pocket of Tus was most effective in arresting the replisome and mutation of C(6) removed the barrier.The polymerase tracking along the template strand traps the C(6) to prevent lock formation; the helicase tracking along the other strand traps the complementary G(6) to aid lock formation.Our results are consistent with the model where strand separation by the helicase unpairs the GC(6) base pair and triggers lock formation immediately before the polymerase can sequester the C(6) base.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Rutgers, the State University of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA pandeyma@rwjms.rutgers.edu.

Show MeSH
Related in: MedlinePlus