uu.seUppsala University Publications
Change search
ReferencesLink to record
Permanent link

Direct link
DNA and protein interactions of mouse polyomavirus large T antigen
Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
1998 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Polyomaviruses require dividing cells to multiply. The regulatory genes express proteins, theT-antigens, necessary for this to occur. Large T-antigen (LT), is a phosphoprotein withmultiple intrinsic activities, e.g. ATPase, helicase, and interaction with cellular factors. Crucial for the regulatory role of LT is its ability to specifically bind DNA containing GRGGC-motifs located in the non-coding regulatory region of the viral genome. This thesis focuses on the characteristics of LT-DNA interactions. Also, it addresses some important questions regarding the role of LT-protein interactions in initiation of DNA synthesis.

The DNA binding specificity of LT was investigated using a binding site selection protocolbased on the polymerase chain reaction. A pool of polynucleotides with randomised sequences was probed with LT. Molecules that bound to LT were selected. All obtained polynucleotides contained tandemly repeated GRGGC-motifs in a head-to-tail arrangement, with a relatively uniform spacing. Experiments with a DNA binding deficient LT produced evidence for an alternative lower affinity binding site.

LT binding to different types of sites was studied using surface plasmon resonance(BIAcore). Both association and dissociation rates were related to motif configuration. LTcomplexed with the origin of replication site was particularly unstable. Comparison betweensites containing one to four motifs in tandem, showed a preference for non-cooperative dimerbinding. Sodium chloride stabilised the complexes. A pH above 7.0 increased dissociationrate.

An enhancer-less polyomavirus (PyV) can be rescued by inserting binding sites for bovinepapillomavirus (BPV) E2 protein, and supplying E2. In BPV, E2 is a transcriptional activatorthat is, together with E1, required for DNA replication. Using a set of E2 mutants, a correlationbetween transcriptional activation and replication of PyV, but not BPV, was found, suggestingdifferent mechanisms for activation. Interaction assays found evidence of a cellular factoracting as a mediator between E2 and LT.

An LT with an amino acid substitution close to the DNA-binding domain could rescueviability in a PyV with a base substitution in the origin of replication. BIAcore analysisshowed no great differences between mutant and wild type interactions. The mutant LT wascomparable to wild type in other selected activities.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 1998. , 38 p.
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 766
Keyword [en]
Biochemistry, T-antigen, DNA-binding, biosensor, enhancer
Keyword [sv]
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Medical Virology
URN: urn:nbn:se:uu:diva-190ISBN: 91-554-4231-5OAI: oai:DiVA.org:uu-190DiVA: diva2:161464
Public defence
1998-05-26, lecture hall B21, Uppsala Biomedical Centre, Uppsala University, Uppsala, 13:15
Available from: 1998-05-05 Created: 1998-05-05Bibliographically approved

Open Access in DiVA

No full text
Buy this publication >>

By organisation
Department of Medical Biochemistry and Microbiology
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Total: 215 hits
ReferencesLink to record
Permanent link

Direct link