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Characterization of the bacteriophage PhiKMV DNA ligase

R Lavigne1, B Roucourt, K Hertveldt

  • 1Laboratory of Gene Technology, Katholieke Universiteit Leuven, Kasteelpark Arenberg 21, Leuven, B-3001, Belgium.

Protein and Peptide Letters
|March 9, 2006
PubMed

Insights

The Pseudomonas aeruginosa bacteriophage phiKMV's gene 17 product (gp17) functions as an ATP-dependent DNA ligase. This enzyme shows potential for molecular biology applications despite some unique DNA-binding characteristics.

Area of Science:

  • Molecular Biology
  • Enzymology
  • Virology

Background:

  • The gene 17 product (gp17) from the bacteriophage phiKMV, which infects Pseudomonas aeruginosa, was investigated.
  • In silico analysis revealed structural similarities between gp17 and T7 DNA ligase.

Purpose of the Study:

  • To determine if gp17 possesses functional DNA ligase activity.
  • To characterize the enzymatic properties of gp17.

Main Methods:

  • In silico analysis for sequence homology.
  • Semi-quantitative activity assays to assess DNA ligase function.
  • Optimization of enzymatic conditions for purified gp17.

Main Results:

  • gp17 was confirmed as a functional, ATP-dependent DNA ligase.
  • Enzymatic activity was optimized at 4°C for 24 hours with sticky-ended double-stranded DNA fragments.
  • The specific activity was determined to be 0.5 Weiss U/μg for the purified His6-tagged gp17.

Conclusions:

  • gp17 is a novel DNA ligase with potential applications in molecular biology.
  • Structural differences in DNA-binding properties compared to T7 DNA ligase warrant further investigation.

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