Role of Disulfide Bridges in Archaeal Family-B DNA Polymerases

التفاصيل البيبلوغرافية
العنوان: Role of Disulfide Bridges in Archaeal Family-B DNA Polymerases
المؤلفون: Bernard A. Connolly, Tom Killelea
المصدر: ChemBioChem. 12:1330-1336
بيانات النشر: Wiley, 2011.
سنة النشر: 2011
مصطلحات موضوعية: Models, Molecular, Protein Folding, DNA polymerase, Molecular Sequence Data, DNA-Directed DNA Polymerase, Polymerase Chain Reaction, Biochemistry, chemistry.chemical_compound, Serine, Amino Acid Sequence, Cysteine, Disulfides, Molecular Biology, Polymerase, Thermostability, Calorimetry, Differential Scanning, biology, Organic Chemistry, Temperature, biology.organism_classification, Hyperthermophile, Thermococcales, DNA, Archaeal, chemistry, biology.protein, Pyrococcus furiosus, Molecular Medicine, Sequence Alignment, DNA
الوصف: The family-B DNA polymerases obtained from the order Thermococcales, for example, Pyrococcus furiosus (Pfu-Pol) are commonly used in the polymerase chain reaction (PCR) because of their high thermostability and low error rates. Most of these polymerases contain four cysteines, arranged as two disulfide bridges. With Pfu-Pol C429-C443 forms one of the disulfides (DB1) and C507-C510 (DB2) the other. Although the disulfides are well conserved in the enzymes from the hyperthermophilic Thermococcales, they are less prevalent in euryarchaeal polymerases from other orders, and tend to be only found in other hyperthermophiles. Here, we report on the effects of deleting the disulfide bridges by mutating the relevant cysteines to serines. A variety of techniques, including differential scanning calorimetry and differential scanning fluorimetry, have shown that both disulfides make a contribution to thermostability, with DB1 being more important than DB2. However, even when both disulfides are removed, sufficient thermostability remains for normal (identical to the wild type) performance in PCR and quantitative (real-time) PCR. Therefore, polymerases totally lacking cysteine are fully compatible with most PCR-based applications. This observation opens the way to further engineering of polymerases by introduction of a single cysteine followed by appropriate chemical modification.
تدمد: 1439-4227
URL الوصول: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::969ebb5feeb928f4d4c27d7210fe2e53
https://doi.org/10.1002/cbic.201100145
حقوق: CLOSED
رقم الأكسشن: edsair.doi.dedup.....969ebb5feeb928f4d4c27d7210fe2e53
قاعدة البيانات: OpenAIRE