Direct comparison of nick-joining activity of the nucleic acid ligases from bacteriophage T4

Bullard, Desmond R. and Bowater, Richard P. ORCID: (2006) Direct comparison of nick-joining activity of the nucleic acid ligases from bacteriophage T4. Biochemical Journal, 398 (1). pp. 135-144. ISSN 0264-6021

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The genome of bacteriophage T4 encodes three polynucleotide ligases, which seal the backbone of nucleic acids during infection of host bacteria. The T4Dnl (T4 DNA ligase) and two RNA ligases [T4Rnl1 (T4 RNA ligase 1) and T4Rnl2] join a diverse array of substrates, including nicks that are present in double-stranded nucleic acids, albeit with different efficiencies. To unravel the biochemical and functional relationship between these proteins, a systematic analysis of their substrate specificity was performed using recombinant proteins. The ability of each protein to ligate 20 bp double-stranded oligonucleotides containing a single-strand break was determined. Between 4 and 37 °C, all proteins ligated substrates containing various combinations of DNA and RNA. The RNA ligases ligated a more diverse set of substrates than T4Dnl and, generally, T4Rnl1 had 50-1000-fold lower activity than T4Rnl2. In assays using identical conditions, optimal ligation of all substrates was at pH 8 for T4Dnl and T4Rnl1 and pH 7 for T4Rnl2, demonstrating that the protein dictates the pH optimum for ligation. All proteins ligated a substrate containing DNA as the unbroken strand, with the nucleotides at the nick of the broken strand being RNA at the 3'-hydroxy group and DNA at the 5'-phosphate. Since this RNA-DNA hybrid was joined at a similar maximal rate by T4Dnl and T4Rnl2 at 37 °C, we consider the possibility that this could be an unexpected physiological substrate used during some pathways of 'DNA repair'.

Item Type: Article
Faculty \ School: Faculty of Science
Faculty of Science > School of Biological Sciences
UEA Research Groups: Faculty of Science > Research Groups > Molecular Microbiology
Faculty of Science > Research Centres > Centre for Molecular and Structural Biochemistry
Depositing User: Vishal Gautam
Date Deposited: 01 May 2006
Last Modified: 08 Feb 2023 17:30
DOI: 10.1042/BJ20060313

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