Limited terminal transferase in human DNA polymerase mu defines the required balance between accuracy and efficiency in NHEJ.
DNA polymerase mu (Polmu) 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 Polmu can be specifically required during non-homologous end joining (NHEJ) to create or increase complementarity of DNA ends. By site-directed mutagenesis in human Polmu, we have identified a specific DNA ligand residue (Arg(387)) that is responsible for its limited terminal transferase activity compared to that of human TdT, its closest homologue (42% amino acid identity). Polmu 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 Arg(387) 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 Polmu 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 Polmu could regulate the balance between accuracy and necessary efficiency, providing some variability during NHEJ.
Andrade
Paula
P
Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, 28049 Madrid, Spain.
Martín
María José
MJ
Juárez
Raquel
R
López de Saro
Francisco
F
Blanco
Luis
L
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage; D004260:DNA Repair; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014166:Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D006639:Histidine; D001120:Arginine; D014166:Transferases; C407743:DNA polymerase mu; D004259:DNA-Directed DNA Polymerase