Source:http://linkedlifedata.com/resource/pubmed/id/11483153
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| Predicate | Object |
|---|---|
| rdf:type | |
| lifeskim:mentions | |
| pubmed:issue |
Pt 1
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| pubmed:dateCreated |
2001-8-2
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| pubmed:databankReference | |
| pubmed:abstractText |
A gene encoding a DNA polymerase I from the thermophilic eubacterium Rhodothermus marinus was identified. The gene was cloned, sequenced and expressed in Escherichia coli. The gene is 2772 bp long and encodes a protein of 924 amino acids with a calculated molecular mass of 104.8 kDa. Sequence analysis showed that a generally conserved Phe residue in the O-helix is substituted by a Tyr (position 756) in the R. marinus enzyme. A Tyr in this position decreases the discrimination against dideoxynucleotides which is a major advantage in DNA sequencing. The protein was purified, characterized and showed to contain specific DNA-polymerization activity of 3100 units/mg of protein, 5'-->3' exonuclease activity and a 3'-->5' proofreading activity. Its optimum activity was at 55 degrees C and it had a half-life of 2 min at 90 degrees C. A truncated form of the enzyme lacking the 5'-->3' exonuclease domain was also expressed in E. coli. It had a specific DNA-polymerization activity of 5000 units/mg of protein and lacked the 5'-->3' exonuclease activity. Its optimum activity was at 65 degrees C and it had a half-life of 11 min at 90 degrees C. It was usable for DNA sequencing. This is the first thermostable DNA polymerase described with the O-helix Phe-->Tyr substitution.
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| pubmed:language |
eng
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| pubmed:journal | |
| pubmed:citationSubset |
IM
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| pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/DNA, Bacterial,
http://linkedlifedata.com/resource/pubmed/chemical/DNA Polymerase I,
http://linkedlifedata.com/resource/pubmed/chemical/Exonucleases,
http://linkedlifedata.com/resource/pubmed/chemical/Protein Isoforms,
http://linkedlifedata.com/resource/pubmed/chemical/RNA-Directed DNA Polymerase
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| pubmed:status |
MEDLINE
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| pubmed:month |
Aug
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| pubmed:issn |
0885-4513
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| pubmed:author | |
| pubmed:issnType |
Print
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| pubmed:volume |
34
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| pubmed:owner |
NLM
|
| pubmed:authorsComplete |
Y
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| pubmed:pagination |
37-45
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| pubmed:dateRevised |
2007-3-21
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| pubmed:meshHeading |
pubmed-meshheading:11483153-Amino Acid Sequence,
pubmed-meshheading:11483153-Bacteria,
pubmed-meshheading:11483153-Cloning, Molecular,
pubmed-meshheading:11483153-DNA, Bacterial,
pubmed-meshheading:11483153-DNA Polymerase I,
pubmed-meshheading:11483153-Escherichia coli,
pubmed-meshheading:11483153-Exonucleases,
pubmed-meshheading:11483153-Hydrogen-Ion Concentration,
pubmed-meshheading:11483153-Molecular Sequence Data,
pubmed-meshheading:11483153-Plasmids,
pubmed-meshheading:11483153-Polymerase Chain Reaction,
pubmed-meshheading:11483153-Protein Isoforms,
pubmed-meshheading:11483153-Protein Structure, Tertiary,
pubmed-meshheading:11483153-RNA-Directed DNA Polymerase,
pubmed-meshheading:11483153-Sequence Analysis, DNA,
pubmed-meshheading:11483153-Sequence Homology, Amino Acid,
pubmed-meshheading:11483153-Time Factors,
pubmed-meshheading:11483153-Transcription, Genetic
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| pubmed:year |
2001
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| pubmed:articleTitle |
Cloning, sequence analysis and functional characterization of DNA polymerase I from the thermophilic eubacterium Rhodothermus marinus.
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| pubmed:affiliation |
Institute of Biology, University of Iceland, Grensásvegur 12, IS-108 Reykjavik, Iceland.
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| pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't
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