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Title: The biochemical and biophysical characterisation of DNA and RNA ligases from bacteriophage T4
Author: Bullard, Desmond.
ISNI:       0000 0001 3508 6180
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2007
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Bacteriophage T4 encodes three polynucleotide ligases that seal phosphodiester backbones during infection of E. coli, referred to as T4Dnl, T4Rnll and T4Rn12. A detailed biochemical examination of the nucleic acid substrate specificity of each ligase was performed using recombinant proteins and a variety of double stranded substrates comprised of a mixture of DNA and RNA, with a single nick. The RNA ligases sealed a broad range of substrates, with the activity of T4Rnll being 50-1000 fold less than that of T4Rn12. Mutagenesis identified regions of the RNA ligases that are important for nick-joining activity. T4Dni had a greater specificity than both RNA ligases, since it was not active on some substrates containing RNA. All proteins joined a double stranded substrate with the 3'-hydroxyl group at the nick being RNA, but with the 5'phosphate group at the nick and the complementary strand being DNA. The conserved use of this substrate questions whether it may be important during nucleic acid metabolism. In assays under identical conditions, the rates of nick-joining by all three recombinant ligases were best at 37°C. For all substrates, optimal1igation was at pH 8.0 for T4Dni and T4Rnll, and pH 7.0 for T4Rn12, therefore showing that the pH optima of ligation is determined by the protein. Changes to buffer conditions showed that all three proteins are dependent upon ATP and Mi+ in order for ligation to occur, although Mn2+ can provide an alternative to Mg2+. Furthermore, Ml+ and ATP along with DTT influenced intrinsic fluorescence emission by the proteins, suggesting that these constituents influence the structure of the proteins. The genome of Streptomyces avermitilis encodes a possible RNA ligase, which was cloned and purified for biochemical analysis. The recombinant protein exhibited weak nick joining activity, although further study is required to provide a better insight into this novel nucleic acid repair process in bacteria.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available