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Visualizing DNA replication at the single-molecule level

Journal Article


Abstract


  • Recent advances in single-molecule methodology have made it possible to study the dynamic behavior of individual enzymes and their interactions with other proteins in multiprotein complexes. Here, we describe newly developed methods to study the coordination of DNA unwinding, priming, and synthesis at the DNA-replication fork. The length of individual DNA molecules is used to measure the activity of single replisomes engaged in coordinated DNA replication. First, a tethered-particle technique is used to visualize the formation and release of replication loops. Second, a fluorescence imaging method provides a direct readout of replication rates and processivities from individual replisomes. The ability to directly observe transient reaction intermediates and characterize heterogeneous behavior makes these single-molecule approaches important new additions to the tools available to study DNA replication.

Publication Date


  • 2010

Citation


  • Tanner, N. A. & van Oijen, A. M. (2010). Visualizing DNA replication at the single-molecule level. Methods in Enzymology, 475 259-278.

Scopus Eid


  • 2-s2.0-77954575187

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/2206

Has Global Citation Frequency


Number Of Pages


  • 19

Start Page


  • 259

End Page


  • 278

Volume


  • 475

Place Of Publication


  • United States

Abstract


  • Recent advances in single-molecule methodology have made it possible to study the dynamic behavior of individual enzymes and their interactions with other proteins in multiprotein complexes. Here, we describe newly developed methods to study the coordination of DNA unwinding, priming, and synthesis at the DNA-replication fork. The length of individual DNA molecules is used to measure the activity of single replisomes engaged in coordinated DNA replication. First, a tethered-particle technique is used to visualize the formation and release of replication loops. Second, a fluorescence imaging method provides a direct readout of replication rates and processivities from individual replisomes. The ability to directly observe transient reaction intermediates and characterize heterogeneous behavior makes these single-molecule approaches important new additions to the tools available to study DNA replication.

Publication Date


  • 2010

Citation


  • Tanner, N. A. & van Oijen, A. M. (2010). Visualizing DNA replication at the single-molecule level. Methods in Enzymology, 475 259-278.

Scopus Eid


  • 2-s2.0-77954575187

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/2206

Has Global Citation Frequency


Number Of Pages


  • 19

Start Page


  • 259

End Page


  • 278

Volume


  • 475

Place Of Publication


  • United States