Real-time single-molecule observation of rolling-circle DNA replication.
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.
Tanner
Nathan A
NA
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Loparo
Joseph J
JJ
Hamdan
Samir M
SM
Jergic
Slobodan
S
Dixon
Nicholas E
NE
van Oijen
Antoine M
AM
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
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