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Chlorella virus DNA ligase: nick recognition and mutational analysis
1Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021, USA.
Nucleic Acids Research
|February 28, 1998
Summary
Chlorella virus DNA ligase uses specific active site residues to seal DNA nicks. Mutational analysis reveals distinct roles for lysine, aspartate, and arginine in DNA binding and catalysis.
Area of Science:
- Biochemistry
- Molecular Biology
- Virology
Background:
- DNA ligases are crucial enzymes that repair DNA breaks.
- Chlorella virus PBCV-1 DNA ligase is a model system for studying DNA ligation mechanisms.
Purpose of the Study:
- To elucidate the functional roles of conserved residues in the KxDGxR active site motif of Chlorella virus PBCV-1 DNA ligase.
- To understand the enzyme's substrate discrimination and catalytic steps.
Main Methods:
- Site-directed mutagenesis of the KxDGxR motif (K27, D29, R32).
- Biochemical assays to assess DNA binding, adenylation, and phosphodiester bond formation.
- Analysis of mutant enzyme activity and substrate interaction.
Main Results:
- Mutant K27A is impaired in initial adenylation but can perform strand closure on pre-adenylated DNA, indicating lysine's role in activation, not closure.
- K27A shows altered DNA binding, suggesting AMP binding pocket occupancy is key for nick recognition.
- Mutant D29A can form the enzyme-adenylate intermediate but not the DNA-adenylate intermediate.
- Mutant R32A is catalytically inactive and shows no DNA binding.
Conclusions:
- The KxDGxR motif residues play distinct, essential roles in DNA ligase activity.
- Lysine (K27) is critical for DNA activation, while its presence is also important for nick recognition.
- Aspartate (D29) is essential for DNA adenylation, and Arginine (R32) is vital for overall catalysis and DNA binding.