Fluorescent single-stranded DNA binding protein as a probe for sensitive, real-time assays of helicase activity.
The formation and maintenance of single-stranded DNA (ssDNA) are essential parts of many processes involving DNA. For example, strand separation of double-stranded DNA (dsDNA) is catalyzed by helicases, and this exposure of the bases on the DNA allows further processing, such as replication, recombination, or repair. Assays of helicase activity and probes for their mechanism are essential for understanding related biological processes. Here we describe the development and use of a fluorescent probe to measure ssDNA formation specifically and in real time, with high sensitivity and time resolution. The reagentless biosensor is based on the ssDNA binding protein (SSB) from Escherichia coli, labeled at a specific site with a coumarin fluorophore. Its use in the study of DNA manipulations involving ssDNA intermediates is demonstrated in assays for DNA unwinding, catalyzed by DNA helicases.
Dillingham
Mark S
MS
DNA:Protein Interactions Unit, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, United Kingdom.
Tibbles
Katherine L
KL
Hunter
Jackie L
JL
Bell
Jason C
JC
Kowalczykowski
Stephen C
SC
Webb
Martin R
MR
D001681:Biological Assay / Q000379:methods*; D003374:Coumarins / Q000378:metabolism; D004265:DNA Helicases / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism*; D007700:Kinetics; D009994:Osmolar Concentration; D013379:Substrate Specificity; D013997:Time Factors; D014364:Tryptophan
D003374:Coumarins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; C476430:SSB protein, E coli; D014364:Tryptophan; D004265:DNA Helicases