Visualizing DNA replication at the single-molecule level.
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.
Tanner
Nathan A
NA
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.
van Oijen
Antoine M
AM
D004247:DNA / Q000737:chemistry*; D004261:DNA Replication; D005898:Glass / Q000737:chemistry; D008856:Microscopy, Fluorescence; D009097:Multienzyme Complexes; D013499:Surface Properties
D009097:Multienzyme Complexes; D004247:DNA