Visualizing DNA replication at the single-molecule level

Authors

Nathan Tanner, Harvard Medical School
Antoine M. van Oijen, Harvard Medical SchoolFollow

RIS ID

94211

Publication Details

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

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.