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Lack of sugar discrimination by human Pol mu requires a single glycine residue

José F Ruiz1, Raquel Juárez, Miguel García-Díaz

  • 1Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Campus de la Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.

Nucleic Acids Research
|July 31, 2003
PubMed

Insights

DNA polymerase mu (Pol mu) poorly discriminates against ribonucleotides, inserting them into DNA chains. A single amino acid change significantly enhanced this discrimination, offering insights into DNA repair mechanisms.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • DNA polymerase mu (Pol mu) is a family X DNA polymerase implicated in DNA repair pathways.
  • Its role in micro-homology mediated joining and double-strand break repair is under investigation.

Purpose of the Study:

  • To investigate the substrate discrimination capabilities of human Pol mu, specifically its ability to differentiate between deoxyribonucleotides (dNTPs) and ribonucleotides (rNTPs).
  • To identify the molecular determinants responsible for sugar discrimination in Pol mu.

Main Methods:

  • Biochemical assays to measure the incorporation efficiency of rNTPs versus dNTPs by Pol mu.
  • 3D structural modeling of human Pol mu based on homologous polymerase structures.
  • Site-directed mutagenesis to alter specific amino acid residues and assess the impact on nucleotide incorporation.

Main Results:

  • Human Pol mu exhibits significantly low discrimination against the 2'-OH group of the sugar moiety, inserting rNTPs with an efficiency only <10-fold lower than dNTPs.
  • Pol mu can incorporate both deoxy- and ribonucleotides into nucleic acid chains and extend both DNA and RNA primers.
  • Mutation of Gly433 to tyrosine in Pol mu resulted in a substantial increase in discrimination against ribonucleotides.

Conclusions:

  • The lack of sugar discrimination by Pol mu is an unusual characteristic for a DNA polymerase.
  • Structural modeling and mutagenesis identified key residues involved in sugar discrimination, suggesting a mechanism for Pol mu's unique substrate preference.
  • These findings provide insights into the potential roles of Pol mu in DNA repair processes where flexibility in nucleotide incorporation may be advantageous.

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