Show simple item record

dc.contributor.authorPiotrowski, Yvonne
dc.contributor.authorGurung, Man Kumari
dc.contributor.authorLarsen, Atle Noralf
dc.date.accessioned2020-02-21T12:10:11Z
dc.date.available2020-02-21T12:10:11Z
dc.date.issued2019-08-09
dc.description.abstract<i>Background</i> - The discovery of thermostable DNA polymerases such as Taq DNA polymerase revolutionized amplification of DNA by polymerase chain reaction methods that rely on thermal cycling for strand separation. These methods are widely used in the laboratory for medical research, clinical diagnostics, criminal forensics and general molecular biology research. Today there is a growing demand for on-site molecular diagnostics; so-called ‘Point-of-Care tests’. Isothermal nucleic acid amplification techniques do not require a thermal cycler making these techniques more suitable for performing Point-of-Care tests at ambient temperatures compared to traditional polymerase chain reaction methods. Strand-displacement activity is essential for such isothermal nucleic acid amplification; however, the selection of DNA polymerases with inherent strand-displacement activity that are capable of performing DNA synthesis at ambient temperatures is currently limited.<p><p> <i>Results</i> - We have characterized the large fragment of a DNA polymerase I originating from the marine psychrophilic bacterium Psychrobacillus sp. The enzyme showed optimal polymerase activity at pH 8–9 and 25–110 mM NaCl/KCl. The polymerase was capable of performing polymerase as well as robust strand-displacement DNA synthesis at ambient temperatures (25–37 °C). Through molecular evolution and screening of thousand variants we have identified a single amino-acid exchange of Asp to Ala at position 422 which induced a 2.5-fold increase in strand-displacement activity of the enzyme.<p> Transferring the mutation of the conserved Asp residue to corresponding thermophilic homologues from Ureibacillus thermosphaericus and Geobacillus stearothermophilus also resulted in a significant increase in the strand-displacement activity of the enzymes.<p><p> <i>Conclusions</i> - Substituting Asp with Ala at positon 422 resulted in a significant increase in strand-displacement activity of three prokaryotic A-family DNA polymerases adapted to different environmental temperatures i.e. being psychrophilic and thermophilic of origin. This strongly indicates an important role for the 422 position and the O1-helix for strand-displacement activity of DNA polymerase I. The D422A variants generated here may be highly useful for isothermal nucleic acid amplification at a wide temperature scale.en_US
dc.identifier.citationPiotrowski Y, Gurung G, Larsen anl. Characterization and engineering of a DNA polymerase reveals a single amino-acid substitution in the fingers subdomain to increase strand-displacement activity of A-family prokaryotic DNA polymerases. BMC Molecular and Cell Biology. 2019en_US
dc.identifier.cristinIDFRIDAID 1716376
dc.identifier.doi10.1186/s12860-019-0216-1
dc.identifier.issn2661-8850
dc.identifier.urihttps://hdl.handle.net/10037/17445
dc.language.isoengen_US
dc.publisherSpringer Natureen_US
dc.relation.journalBMC Molecular and Cell Biology
dc.relation.projectIDinfo:eu-repo/grantAgreement/RCN/BIOTEK2021/226193/Norway/Optimization and application development of marine cold-adapted DNA polymerases/OptiZyme/en_US
dc.rights.accessRightsopenAccessen_US
dc.rights.holderCopyright 2019 The Author(s)en_US
dc.subjectVDP::Medical disciplines: 700::Basic medical, dental and veterinary science disciplines: 710en_US
dc.subjectVDP::Medisinske Fag: 700::Basale medisinske, odontologiske og veterinærmedisinske fag: 710en_US
dc.titleCharacterization and engineering of a DNA polymerase reveals a single amino-acid substitution in the fingers subdomain to increase strand-displacement activity of A-family prokaryotic DNA polymerasesen_US
dc.type.versionpublishedVersionen_US
dc.typeJournal articleen_US
dc.typeTidsskriftartikkelen_US
dc.typePeer revieweden_US


File(s) in this item

Thumbnail

This item appears in the following collection(s)

Show simple item record