Molecular basis for RNA discrimination by human DNA ligase 1

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• DNA ligase 1 (LIG1) is essential for DNA replication and repair, joining DNA nicks.
• LIG1 exhibits high specificity for DNA over DNA-RNA junctions, crucial for preventing mutagenic cycles.
• The precise mechanisms of LIG1's ribonucleotide discrimination remain unclear.

Purpose of the Study:

• To elucidate the structural and kinetic basis of LIG1's discrimination against ribonucleotides.
• To identify the molecular determinants responsible for LIG1's high-fidelity ligation.

Main Methods:

• Structural analysis of LIG1-DNA-RNA complexes.
• Kinetic studies of wild-type and mutant LIG1.
• Site-directed mutagenesis of the identified RNA-binding gate.

Main Results:

• LIG1 utilizes a conserved aromatic steric gate to exclude ribonucleotides from the active site.
• Mutation of the steric gate significantly reduced LIG1's discrimination against RNA substrates (by ~3600-fold).
• The gate plays a critical role in stabilizing the enzyme-substrate complex and ensuring fidelity.

Conclusions:

• The aromatic steric gate is a key determinant of ribonucleotide discrimination in LIG1.
• This mechanism ensures high-fidelity DNA ligation, preventing errors during replication and repair.
• The findings highlight parallels with ribonucleotide discrimination mechanisms in high-fidelity DNA polymerases.