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rdf:type |
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lifeskim:mentions |
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pubmed:issue |
13
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pubmed:dateCreated |
2001-7-2
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pubmed:abstractText |
Measurement of steady-state rates of unwinding of double-stranded oligonucleotides by helicases is hampered due to rapid reannealing of the single-stranded DNA products. Including an oligonucleotide in the reaction mixture which can hybridize with one of the single strands can prevent reannealing. However, helicases bind to single-stranded DNA, therefore the additional oligonucleotide can sequester the enzyme, leading to slower observed rates for unwinding. To circumvent this problem, the oligonucleotide that serves as a trap was replaced with a strand of peptide nucleic acid (PNA). Fluorescence polarization was used to determine that a 15mer PNA strand does not bind to the bacteriophage T4 Dda helicase. Steady-state kinetic parameters of unwinding catalyzed by Dda were determined by using PNA as a trapping strand. The substrate consisted of a partial duplex with 15 nt of single-stranded DNA and 15 bp. In the presence of 250 nM substrate and 1 nM Dda, the rate of unwinding in the presence of the DNA trapping strand was 0.30 nM s(-1) whereas the rate was 1.34 nM s(-1) in the presence of the PNA trapping strand. PNA prevents reannealing of single-stranded DNA products, but does not sequester the helicase. This assay will prove useful in defining the complete kinetic mechanism for unwinding of oligonucleotide substrates by this helicase.
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pubmed:grant |
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pubmed:commentsCorrections |
http://linkedlifedata.com/resource/pubmed/commentcorrection/11433029-10594814,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11433029-10625495,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11433029-10679457,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11433029-11139627,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11433029-11148049,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11433029-1279811,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11433029-1328208,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11433029-2545238,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11433029-6092351,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11433029-6302682,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11433029-7673202,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11433029-7692304,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11433029-7958978,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11433029-8022830,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11433029-8132462,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11433029-8392863,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11433029-8614617,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11433029-8662873,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11433029-8811178,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11433029-8994032,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11433029-9201945,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11433029-9201946,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11433029-9229111,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11433029-9525882,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11433029-9862979,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11433029-9873519
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pubmed:language |
eng
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pubmed:journal |
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pubmed:citationSubset |
IM
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pubmed:chemical |
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pubmed:status |
MEDLINE
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pubmed:month |
Jul
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pubmed:issn |
1362-4962
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pubmed:author |
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pubmed:issnType |
Electronic
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pubmed:day |
1
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pubmed:volume |
29
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
2829-35
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pubmed:dateRevised |
2009-11-18
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pubmed:meshHeading |
pubmed-meshheading:11433029-Adenosine Triphosphate,
pubmed-meshheading:11433029-Bacteriophage T4,
pubmed-meshheading:11433029-Base Pairing,
pubmed-meshheading:11433029-Base Sequence,
pubmed-meshheading:11433029-Catalysis,
pubmed-meshheading:11433029-DNA,
pubmed-meshheading:11433029-DNA, Single-Stranded,
pubmed-meshheading:11433029-DNA Helicases,
pubmed-meshheading:11433029-Fluorescence Polarization,
pubmed-meshheading:11433029-Kinetics,
pubmed-meshheading:11433029-Nucleic Acid Conformation,
pubmed-meshheading:11433029-Nucleic Acid Denaturation,
pubmed-meshheading:11433029-Nucleic Acid Hybridization,
pubmed-meshheading:11433029-Peptide Nucleic Acids,
pubmed-meshheading:11433029-Protein Binding,
pubmed-meshheading:11433029-Viral Proteins
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pubmed:year |
2001
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pubmed:articleTitle |
Measurement of steady-state kinetic parameters for DNA unwinding by the bacteriophage T4 Dda helicase: use of peptide nucleic acids to trap single-stranded DNA products of helicase reactions.
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pubmed:affiliation |
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 West Markham, Little Rock, AR 72205, USA.
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pubmed:publicationType |
Journal Article,
Research Support, U.S. Gov't, P.H.S.
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