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’.
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August 2006
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Research Article|
July 27 2006
Direct comparison of nick-joining activity of the nucleic acid ligases from bacteriophage T4
Desmond R. Bullard;
Desmond R. Bullard
1School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, U.K.
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Richard P. Bowater
Richard P. Bowater
1
1School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, U.K.
1To whom correspondence should be addressed (email r.bowater@uea.ac.uk).
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Biochem J (2006) 398 (1): 135–144.
Article history
Received:
February 24 2006
Revision Received:
May 03 2006
Accepted:
May 04 2006
Accepted Manuscript online:
May 04 2006
Citation
Desmond R. Bullard, Richard P. Bowater; Direct comparison of nick-joining activity of the nucleic acid ligases from bacteriophage T4. Biochem J 15 August 2006; 398 (1): 135–144. doi: https://doi.org/10.1042/BJ20060313
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