Polμ tumor variants decrease the efficiency and accuracy of NHEJ.

The non homologous end-joining (NHEJ) pathway of double-strand break (DSB) repair often requires DNA synthesis to fill the gaps generated upon alignment of the broken ends, a complex task performed in human cells by two specialized DNA polymerases, Pol lambda and Pol mu. It is now well established that Pol mu is the one adapted to repair DSBs with non-complementary ends, the most challenging scenario, although the structural basis and physiological implications of this adaptation are not fully understood. Here, we demonstrate that two human Pol mu point mutations, G174S and R175H, previously identified in two different tumor samples and affecting two adjacent residues, limit the efficiency of accurate NHEJ by Pol mu in vitro and in vivo. Moreover, we show that this limitation is the consequence of a decreased template dependency during NHEJ, which renders the error-rate of the mutants higher due to the ability of Pol mu to randomly incorporate nucleotides at DSBs. These results highlight the relevance of the 8 kDa domain of Pol mu for accurate and efficient NHEJ, but also its contribution to the error-prone behavior of Pol mu at 2-nt gaps. This work provides the first demonstration that mutations affecting Pol mu identified in tumors can alter the efficiency and fidelity of NHEJ.