title: Real-time single-molecule observation of rolling-circle DNA replication.
abstract: We present a simple technique for visualizing replication of individual DNA molecules in real time. By attaching a rolling-circle substrate to a TIRF microscope-mounted flow chamber, we are able to monitor the progression of single-DNA synthesis events and accurately measure rates and processivities of single T7 and Escherichia coli replisomes as they replicate DNA. This method allows for rapid and precise characterization of the kinetics of DNA synthesis and the effects of replication inhibitors.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Nathan A
authors.initials: NA
authors.lastname: Tanner
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Loparo
authors.forename: Samir M
authors.initials: SM
authors.lastname: Hamdan
authors.forename: Slobodan
authors.initials: S
authors.lastname: Jergic
authors.forename: Nicholas E
authors.initials: NE
authors.lastname: Dixon
authors.forename: Antoine M
authors.initials: AM
authors.lastname: van Oijen
mesh_terms: D017123:Bacteriophage T7 / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication / Q000187:drug effects; D004269:DNA, Bacterial / Q000096:biosynthesis; D004279:DNA, Viral / Q000096:biosynthesis; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000494:pharmacology; D054306:Dideoxynucleotides / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods*; D009097:Multienzyme Complexes / Q000378:metabolism; D009930:Organic Chemicals / Q000737:chemistry; D000099272:Replisomes
chemical_list: D004269:DNA, Bacterial; D004279:DNA, Viral; D004259:DNA-Directed DNA Polymerase; D003848:Deoxyguanine Nucleotides; D054306:Dideoxynucleotides; D005456:Fluorescent Dyes; D009097:Multienzyme Complexes; D009930:Organic Chemicals; D000099272:Replisomes; C416802:SYTOX Orange dye; C031709:2',3'-dideoxyguanosine 5'-triphosphate