当前位置： SCI文献检索 > INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES期刊下所有文献 > Characterization and application of a family B DNA polymerase from the hyperthermophilic and radioresistant euryarchaeon Thermococcus gammatolerans.

Characterization and application of a family B DNA polymerase from the hyperthermophilic and radioresistant euryarchaeon Thermococcus gammatolerans.

Abstract：

:Thermococcus gammatolerans is anaerobic euryarchaeon which grows optimally at 88 °C and its genome encodes a family B DNA polymerase (Tga PolB). Herein, we cloned the gene of Tga PolB, expressed and purified the gene product, and characterized the enzyme biochemically. The recombinant Tga PolB can efficiently synthesize DNA at high temperature, and retain 93% activity after heated at 95 °C for 1.0 h, suggesting that the enzyme is thermostable. Furthermore, the optimal pH for the enzyme activity was measured to be 7.0-9.0. Tga PolB activity is dependent on a divalent cation, among which magnesium ion is optimal. NaCl at low concentration stimulates the enzyme activity but at high concentration inhibits enzyme activity. Interestingly, Tga PolB is able to efficiently bypass uracil in DNA, which is distinct from other archaeal family B DNA pols. By contrast, Tga PolB is halted by an AP site in DNA, as observed in other archaeal family B DNA polymerases. Furthermore, Tga PolB extends the mismatched ends with reduced efficiencies. The enzyme possesses 3'-5' exonuclease activity and this activity is inhibited by dNTPs. The DNA binding assays showed that Tga PolB can efficiently bind to ssDNA and primed DNA, and have a marked preference for primed DNA. Last, Tga PolB can be used in routine PCR.

journal_name

Int J Biol Macromol

journal_title

International journal of biological macromolecules

authors

Zhang L,Jiang D,Shi H,Wu M,Gan Q,Yang Z,Oger P

doi

10.1016/j.ijbiomac.2020.03.204

subject

Has Abstract

pub_date

2020-08-01 00:00:00

pages

217-224

eissn

0141-8130

issn

1879-0003

pii

S0141-8130(20)32790-2

journal_volume

156

pub_type

杂志文章

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