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

DNA Polymerase mu (Pol mu) is a novel family X DNA Polymerase that has been suggested to play a role in micro-homology mediated joining and repair of double strand breaks. We show here that human Pol mu is not able to discriminate against the 2'-OH group of the sugar moiety. It inserts rNTPs with an efficiency that is <10-fold lower than that of dNTPs, in sharp contrast with the >1000-fold discrimination characteristic of most DNA-dependent DNA polymerases. The lack of sugar discrimination by Pol mu is demonstrated by its ability to add rNTPs to both DNA and RNA primer strands, and to insert both deoxy- and ribonucleotides on growing nucleic acid chains. 3D-modelling of human Pol mu based on the available Pol beta and TdT structural information allowed us to predict candidate residues involved in sugar discrimination. Thus, a single amino acid substitution in which Gly433 residue of Pol mu was mutated to the consensus tyrosine present in Pol beta, produced a strong increase in the discrimination against ribonucleotides. The unusual capacity to insert both rNTPs and dNTPs will be discussed in the context of the predicted roles of Pol mu in DNA repair.