Supercoiling, Knotting and Replication Fork Reversal in Partially Replicated Plasmids

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2002 Jan 26

PMID 11809877

Citations 32

Authors

L Olavarrieta

M L Martinez-Robles

J M Sogo

A Stasiak

P Hernandez

D B Krimer

J B Schvartzman

Affiliations

Soon will be listed here.

Abstract

To study the structure of partially replicated plasmids, we cloned the Escherichia coli polar replication terminator TerE in its active orientation at different locations in the ColE1 vector pBR18. The resulting plasmids, pBR18-TerE@StyI and pBR18-TerE@EcoRI, were analyzed by neutral/neutral two-dimensional agarose gel electrophoresis and electron microscopy. Replication forks stop at the Ter-TUS complex, leading to the accumulation of specific replication intermediates with a mass 1.26 times the mass of non-replicating plasmids for pBR18-TerE@StyI and 1.57 times for pBR18-TerE@EcoRI. The number of knotted bubbles detected after digestion with ScaI and the number and electrophoretic mobility of undigested partially replicated topoisomers reflect the changes in plasmid topology that occur in DNA molecules replicated to different extents. Exposure to increasing concentrations of chloroquine or ethidium bromide revealed that partially replicated topoisomers (CCCRIs) do not sustain positive supercoiling as efficiently as their non-replicating counterparts. It was suggested that this occurs because in partially replicated plasmids a positive DeltaLk is absorbed by regression of the replication fork. Indeed, we showed by electron microscopy that, at least in the presence of chloroquine, some of the CCCRIs of pBR18-Ter@StyI formed Holliday-like junction structures characteristic of reversed forks. However, not all the positive supercoiling was absorbed by fork reversal in the presence of high concentrations of ethidium bromide.

Citing Articles

Concise Overview of Methodologies Employed in the Study of Bacterial DNA Replication.

Maciag-Dorszynska M, Morcinek-Orlowska J, Baranska S Int J Mol Sci. 2025; 26(2).

PMID: 39859162 PMC: 11764726. DOI: 10.3390/ijms26020446.

Two-Dimensional Gel Electrophoresis to Study the Activity of Type IIA Topoisomerases on Plasmid Replication Intermediates.

Cebrian J, Martinez V, Hernandez P, Krimer D, Fernandez-Nestosa M, Schvartzman J Biology (Basel). 2021; 10(11).

PMID: 34827187 PMC: 8615216. DOI: 10.3390/biology10111195.

Characterization of Episomal Replication of Bovine Papillomavirus Type 1 DNA in Long-Term Virion-Infected Saccharomyces Cerevisiae Culture.

Tu Q, Feng W, Chen Z, Li Q, Zhao Y, Chen J Virol Sin. 2021; 36(6):1492-1502.

PMID: 34460066 PMC: 8692549. DOI: 10.1007/s12250-021-00439-y.

DNA-Topology Simplification by Topoisomerases.

Hanke A, Ziraldo R, Levene S Molecules. 2021; 26(11).

PMID: 34204901 PMC: 8199745. DOI: 10.3390/molecules26113375.

Chromatin Is Frequently Unknotted at the Megabase Scale.

Goundaroulis D, Aiden E, Stasiak A Biophys J. 2019; 118(9):2268-2279.

PMID: 31818464 PMC: 7202934. DOI: 10.1016/j.bpj.2019.11.002.

References

Sundin O, Varshavsky A . Terminal stages of SV40 DNA replication proceed via multiply intertwined catenated dimers. Cell. 1980; 21(1):103-14. DOI: 10.1016/0092-8674(80)90118-x. View

Higgins N, Kato K, Strauss B . A model for replication repair in mammalian cells. J Mol Biol. 1976; 101(3):417-25. DOI: 10.1016/0022-2836(76)90156-x. View

Bell L, Byers B . Separation of branched from linear DNA by two-dimensional gel electrophoresis. Anal Biochem. 1983; 130(2):527-35. DOI: 10.1016/0003-2697(83)90628-0. View

Shishido K, Komiyama N, Ikawa S . Increased production of a knotted form of plasmid pBR322 DNA in Escherichia coli DNA topoisomerase mutants. J Mol Biol. 1987; 195(1):215-8. DOI: 10.1016/0022-2836(87)90338-x. View

Brewer B, FANGMAN W . The localization of replication origins on ARS plasmids in S. cerevisiae. Cell. 1987; 51(3):463-71. DOI: 10.1016/0092-8674(87)90642-8. View

Dasgupta S, Masukata H, Tomizawa J . Multiple mechanisms for initiation of ColE1 DNA replication: DNA synthesis in the presence and absence of ribonuclease H. Cell. 1987; 51(6):1113-22. DOI: 10.1016/0092-8674(87)90597-6. View

Hill T, Pelletier A, Tecklenburg M, KUEMPEL P . Identification of the DNA sequence from the E. coli terminus region that halts replication forks. Cell. 1988; 55(3):459-66. DOI: 10.1016/0092-8674(88)90032-3. View

Khatri G, MacAllister T, Sista P, Bastia D . The replication terminator protein of E. coli is a DNA sequence-specific contra-helicase. Cell. 1989; 59(4):667-74. DOI: 10.1016/0092-8674(89)90012-3. View

Shishido K, Ishii S, Komiyama N . The presence of the region on pBR322 that encodes resistance to tetracycline is responsible for high levels of plasmid DNA knotting in Escherichia coli DNA topoisomerase I deletion mutant. Nucleic Acids Res. 1989; 17(23):9749-59. PMC: 335211. DOI: 10.1093/nar/17.23.9749. View

10.

Schvartzman J, Adolph S, Martin-Parras L, SCHILDKRAUT C . Evidence that replication initiates at only some of the potential origins in each oligomeric form of bovine papillomavirus type 1 DNA. Mol Cell Biol. 1990; 10(6):3078-86. PMC: 360672. DOI: 10.1128/mcb.10.6.3078-3086.1990. View

11.

Martin-Parras L, Hernandez P, Martinez-Robles M, Schvartzman J . Unidirectional replication as visualized by two-dimensional agarose gel electrophoresis. J Mol Biol. 1991; 220(4):843-53. DOI: 10.1016/0022-2836(91)90357-c. View

12.

Gottlieb P, Wu S, Zhang X, Tecklenburg M, Kuempel P, Hill T . Equilibrium, kinetic, and footprinting studies of the Tus-Ter protein-DNA interaction. J Biol Chem. 1992; 267(11):7434-43. View

13.

Martin-Parras L, Hernandez P, Martinez-Robles M, Schvartzman J . Initiation of DNA replication in ColE1 plasmids containing multiple potential origins of replication. J Biol Chem. 1992; 267(31):22496-505. View

14.

Friedman K, Brewer B . Analysis of replication intermediates by two-dimensional agarose gel electrophoresis. Methods Enzymol. 1995; 262:613-27. DOI: 10.1016/0076-6879(95)62048-6. View

15.

Viguera E, Hernandez P, Krimer D, Boistov A, Lurz R, Alonso J . The ColE1 unidirectional origin acts as a polar replication fork pausing site. J Biol Chem. 1996; 271(37):22414-21. DOI: 10.1074/jbc.271.37.22414. View

16.

Krabbe M, Zabielski J, Bernander R, Nordstrom K . Inactivation of the replication-termination system affects the replication mode and causes unstable maintenance of plasmid R1. Mol Microbiol. 1997; 24(4):723-35. DOI: 10.1046/j.1365-2958.1997.3791747.x. View

17.

Viguera E, Hernandez P, Krimer D, Lurz R, Schvartzman J . Visualisation of plasmid replication intermediates containing reversed forks. Nucleic Acids Res. 1999; 28(2):498-503. PMC: 102505. DOI: 10.1093/nar/28.2.498. View

18.

Mohanty B, Sahoo T, Bastia D . Mechanistic studies on the impact of transcription on sequence-specific termination of DNA replication and vice versa. J Biol Chem. 1998; 273(5):3051-9. DOI: 10.1074/jbc.273.5.3051. View

19.

Paulsson J, Ehrenberg M . Trade-off between segregational stability and metabolic burden: a mathematical model of plasmid ColE1 replication control. J Mol Biol. 1998; 279(1):73-88. DOI: 10.1006/jmbi.1998.1751. View

20.

Martin-Parras L, Lucas I, Martinez-Robles M, Hernandez P, Krimer D, Hyrien O . Topological complexity of different populations of pBR322 as visualized by two-dimensional agarose gel electrophoresis. Nucleic Acids Res. 1998; 26(14):3424-32. PMC: 147708. DOI: 10.1093/nar/26.14.3424. View