RecQ Helicase-catalyzed DNA Unwinding Detected by Fluorescence Resonance Energy Transfer
نویسندگان
چکیده
منابع مشابه
Single-turnover kinetics of helicase-catalyzed DNA unwinding monitored continuously by fluorescence energy transfer.
We describe a fluorescence assay that can be used to monitor helicase-catalyzed unwinding of duplex DNA continuously in real time. The assay is based on the observation that fluorescence resonance energy transfer (FRET) occurs between donor (fluorescein) and acceptor (hexachlorofluorescein) fluorophores that are in close proximity due to their covalent attachment to the 3' and 5' ends of the co...
متن کاملMechanisms of helicase-catalyzed DNA unwinding.
DNA helicases are essential motor proteins that function to unwind duplex DNA to yield the transient single-stranded DNA intermediates required for replication, recombination, and repair. These enzymes unwind duplex DNA and translocate along DNA in reactions that are coupled to the binding and hydrolysis of 5'-nucleoside triphosphates (NTP). Although these enzymes are essential for DNA metaboli...
متن کاملChanges in DNA bending and flexing due to tethered cations detected by fluorescence resonance energy transfer
Local DNA deformation arises from an interplay among sequence-related base stacking, intrastrand phosphate repulsion, and counterion and water distribution, which is further complicated by the approach and binding of a protein. The role of electrostatics in this complex chemistry was investigated using tethered cationic groups that mimic proximate side chains. A DNA duplex was modified with one...
متن کاملFluorescence stopped-flow studies of single turnover kinetics of E.coli RecBCD helicase-catalyzed DNA unwinding.
We have developed and optimized a stopped-flow fluorescence assay for use in studying DNA unwinding catalyzed by Escherichia coli RecBCD helicase. This assay monitors changes in fluorescence resonance energy transfer (FRET) between a pair of fluorescent probes (Cy3 donor and Cy5 acceptor) placed on opposite sides of a nick in duplex DNA. As such, this is an "all-or-none" DNA unwinding assay. Si...
متن کامل[13] Fluorescence Resonance Energy Transfer
of the extent of binding. This effect (known informally as "Chip dip") is illustrated in Figs. 6 and 7 which concern the ethidium bromide/tRNA system. 41 In this case the free ethidium bromide has a lifetime of 1.86 nsec and a rotational relaxation time of 0.54 nsec. On binding the PhetRNA Phe the lifetime increases to 26 nsec and the rotational relaxation increases to 136 nsec. A single rotati...
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ژورنال
عنوان ژورنال: Acta Biochimica et Biophysica Sinica
سال: 2005
ISSN: 1672-9145,1745-7270
DOI: 10.1111/j.1745-7270.2005.00084.x