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Characterization of proteolytic fragments of bacteriophage T7 DNA ligase
A J Doherty1, S R Ashford, D B Wigley
1Laboratory of Molecular Biophysics, University of Oxford, UK.
Abstract:
Treatment of T7 DNA ligase with a range of proteases generates two major fragments which are resistant to further digestion. These fragments, of molecular weight 16 and 26 kDa, are derived from the N- and C-termini of the protein, respectively. The presence of ATP or a non-hydrolysable analogue, ADPNP, during limited proteolysis greatly reduces the level of digestion. The N-terminal 16 kDa region of the intact T7 ligase is labelled selectively in the presence of [alpha-32P]ATP, confirming that it contains the active site lysine residue. In common with the intact enzyme, the C-terminal portion of the protein retains the ability to band shift DNA fragments of various lengths, implicating it in DNA binding. It can also inhibit ligation by the intact protein, apparently by competing for target sites on DNA. We conclude that the N-terminal region, which contains the putative active site lysine, plays a role in the transfer of AMP from the enzyme-adenylate complex to the 5'phosphate at the nick site, while the C-terminal 26 kDa fragment appears to position the enzyme at the target site on DNA.
Insights
T7 DNA ligase digestion reveals two stable fragments. The N-terminal fragment contains the active site, while the C-terminal fragment binds DNA, suggesting distinct functional roles in ligation.
Area of Science:
- Molecular Biology
- Enzymology
- Protein Chemistry
Background:
- T7 DNA ligase is crucial for DNA replication and repair.
- Understanding the functional domains of T7 DNA ligase is essential for its application in molecular biology.
Purpose of the Study:
- To investigate the structural and functional domains of T7 DNA ligase.
- To identify the regions responsible for catalytic activity and DNA binding.
Main Methods:
- Limited proteolysis of T7 DNA ligase.
- Analysis of resulting protein fragments by molecular weight.
- Site-directed labeling with [alpha-32P]ATP.
- DNA binding assays (band shift).
Main Results:
- Protease treatment yielded 16 kDa (N-terminal) and 26 kDa (C-terminal) fragments.
- ATP or ADPNP protected the enzyme from digestion.
- The N-terminal fragment contains the active site lysine, confirmed by [alpha-32P]ATP labeling.
- The C-terminal fragment binds DNA and inhibits ligation, suggesting a DNA-targeting role.
Conclusions:
- The N-terminal domain of T7 DNA ligase is involved in the catalytic transfer of AMP.
- The C-terminal domain functions in DNA binding and enzyme positioning.
- These distinct domains contribute to the overall function of T7 DNA ligase in DNA repair.