Limited terminal transferase in human DNA polymerase μ defines the required balance between accuracy and efficiency in NHEJ

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 106; no. 38; pp. 16203 - 16208
• Main Authors: Andrade, Paula, Martín, María José, Juárez, Raquel, López de Saro, Francisco, Blanco, Luis
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 22.09.2009; National Acad Sciences
• Subjects: Amino Acid Sequence; Amino Acid Substitution; Arginine - genetics; Arginine - metabolism; Autoradiography; Biological Sciences; Catalysis; Catalytic Domain - genetics; Crystal structure; DNA; DNA Breaks, Double-Stranded; DNA Damage; DNA Repair; DNA-Directed DNA Polymerase - chemistry; DNA-Directed DNA Polymerase - genetics; DNA-Directed DNA Polymerase - metabolism; Enzymes; Gene expression regulation; Histidine - genetics; Histidine - metabolism; Humans; Mice; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Nucleotides; Oligonucleotides; Polymerization; Protein Binding; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Substrate Specificity; Transferases - chemistry; Transferases - genetics; Transferases - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0908492106

DNA polymerase mu (Polμ) is a family X member implicated in DNA repair, with template-directed and terminal transferase (template-independent) activities. It has been proposed that the terminal transferase activity of Polμ can be specifically required during non-homologous end joining (NHEJ) to create or increase complementarity of DNA ends. By site-directed mutagenesis in human Polμ, we have identified a specific DNA ligand residue (Arg³⁸⁷) that is responsible for its limited terminal transferase activity compared to that of human TdT, its closest homologue (42% amino acid identity). Polμ mutant R387K (mimicking TdT) displayed a large increase in terminal transferase activity, but a weakened interaction with ssDNA. That paradox can be explained by the regulatory role of Arg³⁸⁷ in the translocation of the primer from a non-productive E:DNA complex to a productive E:DNA:dNTP complex in the absence of a templating base, which is probably the rate limiting step during template-independent synthesis. Further, we show that the Polμ switch from terminal transferase to templated insertions in NHEJ reactions is triggered by recognition of a 5'-P at a second DNA end, whose 3'-protrusion could provide a templating base to facilitate such a special "pre-catalytic translocation step." These studies shed light on the mechanism by which a rate-limited terminal transferase activity in Polμ could regulate the balance between accuracy and necessary efficiency, providing some variability during NHEJ.