Compartmentalized self-replication (CSR) selection of Thermococcus litoralis Sh1B DNA polymerase for diminished uracil binding.
The thermostable archaeal DNA polymerase Sh1B from Thermococcus litoralis has a typical uracil-binding pocket, which in nature plays an essential role in preventing the accumulation of mutations caused by cytosine deamination to uracil and subsequent G-C base pair transition to A-T during the genomic DNA replication. The uracil-binding pocket recognizes and binds uracil base in a template strand trapping the polymerase. Since DNA replication stops, the repair systems have a chance to correct the promutagenic event. Archaeal family B DNA polymerases are employed in various PCR applications. Contrary to nature, in PCR the uracil-binding property of archaeal polymerases is disadvantageous and results in decreased DNA amplification yields and lowered sensitivity. Furthermore, in diagnostics qPCR, RT-qPCR and end-point PCR are performed using dNTP mixtures, where dTTP is partially or fully replaced by dUTP. Uracil-DNA glycosylase treatment and subsequent heating of the samples is used to degrade the DNA containing uracil and prevent carryover contamination, which is the main concern in diagnostic laboratories. A thermostable archaeal DNA polymerase with the abolished uracil binding would be a highly desirable and commercially interesting product. An attempt to disable uracil binding in DNA polymerase Sh1B from T. litoralis by generating site-specific mutants did not yield satisfactory results. However, a combination of random mutagenesis of the whole polymerase gene and compartmentalized self-replication was successfully used to select variants of thermostable Sh1B polymerase capable of performing PCR with dUTP instead of dTTP.
R&D, Fermentas UAB, Graiciuno 8, Vilnius LT-02241, Lithuania.
Agne
A
Tubeleviciute
Remigijus
R
Skirgaila
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D019020:Directed Molecular Evolution / Q000379:methods*; D004587:Electrophoresis, Agar Gel; D004795:Enzyme Stability; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D054730:Protein Interaction Domains and Motifs / Q000235:genetics*; D019712:Thermococcus / Q000201:enzymology*; D014498:Uracil / Q000737:chemistry / Q000378:metabolism*
D003856:Deoxyuracil Nucleotides; D014498:Uracil; D004259:DNA-Directed DNA Polymerase