DNA replication is a key process required by organisms during cell division, with a concomitant requirement for genome synthesis by DNA polymerases. Biotechnological exploitation of thermostable DNA polymerases for DNA amplification by the Polymerase Chain Reaction (PCR), provides a significant market for novel enzymes or those with improved properties. An approach was taken to isolate alternative thermostable DNA polymerases, by enriching thermophilic bacteria from a novel thermal environment, aerobically spoiling silage. In addition, a novel DNA polymerase (Abr polBl) was cloned from the thermoacidophilic archaeon, Acidianus brierleyi, with the intention of characterising its in vivo role and application to PCR. Protein sequence analysis suggested a proofreading (high fidelity) DNA synthesis activity most related to polBl DNA polymerases from Crenarchaeota. Abr polBl was heterologously expressed in bacteria and protein purified to homogeneity. Biochemical assays confirmed high-temperature DNA polymerase and 3'-5'exonuclease activities of Abr polBl, with an accompanying proofreading ability. Sequence analysis, processivity, strand displacement and lesion bypass activities indicated potential roles in genome replication and DNA repair. Abr polBl could not amplify DNA under a range of PCR conditions, presumably following its low intrinsic thermostability. Biophysical analyses confirmed irreversible unfolding of Abr polBl at temperatures required for PCR. Supplementation with organic compounds and ionic salts stabilised Abr polBl, promoting retention of conformational stability and DNA synthesis activity following thermal incubation, but could not promote DNA amplification with Abr polB 1.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:589621 |
| Date | January 2012 |
| Creators | Cooper, Christopher D. O. |
| Contributors | Knowles, Chris ; Bailey, Mark ; Louwrier, Ariel ; Thompson, Ian ; Gileadi, Opher |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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