Studies of multifunctional DNA polymerase I from the extremely radiation resistant Deinococcus radiodurans: Recombinant expression, purification and characterization of the full-length protein and its large fragment

A. Fernandes, Y. Piotrowski, A. Williamson, K. Frade, E. Moe

Research output: Contribution to journalArticlepeer-review

Abstract

Deinococcus radiodurans is a bacterium with extreme resistance to desiccation and radiation. Although the origins of this extreme resistance have not been fully elucidated, an efficient DNA repair machinery that includes the enzyme DNA polymerase I, is potentially crucial as part of a protection mechanism. Here we have cloned and performed small, medium, and large-scale expression of full-length D. radiodurans DNA polymerase I (DrPolI) as well as the large/Klenow fragment (DrKlenow). We then carried out functional characterization of 5′ exonuclease, DNA strand displacement and polymerase activities of these proteins using gel-based and molecular beacon-based biochemical assays. With the same expression and purification strategy, we got higher yield in the production of DrKlenow than of the full-length protein, approximately 2.5 mg per liter of culture. Moreover, we detected a prominent 5′ exonuclease activity of DrPolI in vitro. This activity and, DrKlenow strand displacement and DNA polymerase activities are preferentially stimulated at pH 8.0–8.5 and are reduced by addition of NaCl. Interestingly, both protein variants are more thermostable at pH 6.0–6.5. The characterization of DrPolI's multiple functions provides new insights into the enzyme's role in DNA repair pathways, and how the modulation of these functions is potentially used by D. radiodurans as a survival strategy.

Original languageEnglish
Article number105925
JournalProtein Expression And Purification
Volume187
DOIs
Publication statusPublished - Nov 2021

Keywords

  • 5′ exonuclease
  • Deinococcus radiodurans
  • DNA polymerase I
  • DNA strand displacement
  • Recombinant protein production
  • Thermostability

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