Mechanisms Ensuring Fidelity of Family X DNA Polymerases in Programmed DNA rearrangements in Paramecium tetraurelia

Repairing programmed DNA double-strand breaks (DSBs) is crucial in the lifecycle of Paramecium tetraurelia , especially during its sexual reproduction phase when its somatic highly polyploid macronucleus is lost. The formation of a new macronucleus involves Programmed Genome Rearrangements, introducing DNA DSBs at approximately 45,000 loci. P. tetraurelia employs a Non-Homologous End Joining (NHEJ)-related mechanism for the systematic repair of these DSBs. Four genes encoding DNA polymerases of family X are present in the genome, one of which was found recently to colocalize with other proteins of NHEJ. The question arises as to how they make almost no error. Here we show that these enzymes are most similar to metazoan DNA polymerase λ and exhibit high fidelity through two different molecular mechanisms. Using X-ray structure determination of polymerase lambda mutants recapitulating sequence determinants of P. tetraurelia PolXs, we find both a local conformational change that involves exchanging partners in a crucial salt bridge in the active site upon binding of correct dNTPs, and a larger conformational change involving the closure of Loop3. This stabilizes the template DNA in the active site, only in the presence of the correct incoming dNTP. Differences with human pol λ and pol β are discussed.