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Related Experiment Videos

Nick recognition by DNA ligases.

A J Doherty1, T R Dafforn

  • 1Structural Medicine Unit Wellcome Trust Centre for the Study of Molecular Mechanisms in Disease Cambridge Institute for Medical Research, and Department of Haematology, University of Cambridge, Hills Rd, Cambridge, CB2 2XY, UK. aidan@mrc-lmb.cam.ac.uk

Journal of Molecular Biology
|February 5, 2000
PubMed
Summary
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Phage T7 DNA ligase preferentially binds to phosphorylated DNA nicks, not hydroxylated ones, and binds as a monomer. Key lysine residues are essential for both nucleotide binding and nick recognition during DNA sealing.

Area of Science:

  • Molecular Biology
  • Enzymology
  • Structural Biology

Background:

  • DNA ligases are crucial enzymes that seal breaks in DNA.
  • ATP-dependent DNA ligases, like T7 ligase, utilize ATP for their function.
  • The precise mechanism of nick recognition by DNA ligases remains poorly understood.

Purpose of the Study:

  • To investigate the nick recognition mechanism of phage T7 DNA ligase.
  • To identify the specific DNA features recognized by T7 DNA ligase at nicks.
  • To elucidate the structural basis of T7 DNA ligase-DNA interaction and catalytic activity.

Main Methods:

  • DNA nick binding assays comparing phosphorylated vs. hydroxylated nicks.
  • DNA footprinting to determine T7 ligase binding site size and stoichiometry.

Related Experiment Videos

  • Molecular modeling to propose a structural model of the ligase-DNA complex.
  • Photo-crosslinking and site-directed mutagenesis to identify critical residues.

    • Main Results:

    • T7 DNA ligase distinguishes between 5'-phosphate and 5'-OH nicks, preferring phosphorylated nicks.
    • The enzyme binds asymmetrically to nicks as a monomer, covering 12-14 bp.
    • Lysine residues K238 and K240 were identified as critical for transadenylation and nick sealing.
    • Structural analysis suggests these lysines are involved in nucleotide and DNA binding.

    Conclusions:

    • T7 DNA ligase exhibits specific nick recognition based on the 5' end modification.
    • The enzyme functions as a monomer with asymmetric DNA binding.
    • Key lysine residues play a vital role in the catalytic mechanism and DNA recognition, providing insights into DNA ligation.