DNA unwinding assay using streptavidin-bound oligonucleotides.
BACKGROUND
Helicases play essential roles in many cellular processes including replication, transcription and translation. Most helicases translocate along one strand of the duplex while displacing the complementary strand (of either DNA or RNA). Thus, helicases have directionality. They move along nucleic acids in either the 3'--> 5' or 5'--> 3' direction. The directionality of helicases with low activity or of those that cannot initiate duplex unwinding from a substrate that contains only one single-stranded overhang region is difficult to determine.


RESULTS
An improved assay to determine helicase directionality was developed that uses a substrate containing biotinylated oligonucleotides. As a proof of concept, it was shown that the substrates substantially improve helicase activity and directionality determination for several DNA helicases in comparison to more traditional substrates. In addition, a universal substrate that can be used to determine the directionality of both 3'--> 5' and 5'--> 3' helicases was developed.


CONCLUSIONS
It is shown here that the use of a biotin-streptavidin complex as a helicase substrate improves helicase activity and the determination of helicase directionality. The method described is simpler that the currently available techniques.
Shin
Jae-Ho
JH
University of Maryland Biotechnology Institute, Center for Advanced Research in Biotechnology, 9600 Gudelsky Drive, Rockville, MD 20850, USA. shin@umbi.umd.edu <shin@umbi.umd.edu>
Kelman
Zvi
Z
D001426:Bacterial Proteins; D001710:Biotin / Q000031:analogs & derivatives*; D019921:Biotinylation; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism*; D017013:Methanobacteriaceae / Q000201:enzymology; D009841:Oligonucleotides / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000201:enzymology; D013822:Thermoplasma / Q000201:enzymology
D001426:Bacterial Proteins; D009841:Oligonucleotides; C066114:biotin-streptavidin complex; D001710:Biotin; D004265:DNA Helicases