Nucleotides Linkage Analysis of RecBCD DNA Helicase
نویسندگان
چکیده
منابع مشابه
RecBCD Helicase/Nuclease
The Escherichia coli RecBCD enzyme is a multifunctional protein complex (330 kDa) containing three subunits, the products of the recB, recC, and recD genes. This enzyme displays four distinct activities: nuclease, helicase,ATPase, and site-specific recognition of the DNA regulatory sequence chi (crossover hotspot instigator, w). Originally identified as exonuclease V, the RecBCD enzyme is respo...
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...
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A fluorescence assay was used to measure the processivity of Escherichia coli recBCD enzyme helicase activity. Under standard conditions, recBCD enzyme unwinds an average of 30 +/- 3.2 kilobase pairs (kb)/DNA end before dissociating. The average processivity (P obs) of DNA unwinding under these conditions is 0.99997, indicating that the probability of unwinding another base pair is 30,000-fold ...
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RecBCD enzyme is a heterotrimeric helicase/nuclease that initiates homologous recombination at double-stranded DNA breaks. Several of its activities are regulated by the DNA sequence chi (5'-GCTGGTGG-3'), which is recognized in cis by the translocating enzyme. When RecBCD enzyme encounters chi, the intensity and polarity of its nuclease activity are changed, and the enzyme gains the ability to ...
متن کاملCharacterization of the helicase activity of the Escherichia coli RecBCD enzyme using a novel helicase assay.
We describe an assay to measure the extent of enzymatic unwinding of DNA by a DNA helicase. This assay takes advantage of the quenching of the intrinsic protein fluorescence of Escherichia coli SSB protein upon binding to ssDNA and is used to characterize the DNA unwinding activity of recBCD enzyme. Unwinding in this assay is dependent on the presence of recBCD enzyme and linear dsDNA, is consi...
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ژورنال
عنوان ژورنال: Biophysical Journal
سال: 2015
ISSN: 0006-3495
DOI: 10.1016/j.bpj.2014.11.421