RESUMO
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, Polλ and Polµ. It is now well established that Polµ 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 Polµ point mutations, G174S and R175H, previously identified in two different tumor samples and affecting two adjacent residues, limit the efficiency of accurate NHEJ by Polµ 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 Polµ to randomly incorporate nucleotides at DSBs. These results highlight the relevance of the 8 kDa domain of Polµ for accurate and efficient NHEJ, but also its contribution to the error-prone behavior of Polµ at 2-nt gaps. This work provides the first demonstration that mutations affecting Polµ identified in tumors can alter the efficiency and fidelity of NHEJ.
Assuntos
Reparo do DNA por Junção de Extremidades/genética , DNA Polimerase Dirigida por DNA/genética , Mutagênese/fisiologia , Mutação de Sentido Incorreto , Mutação Puntual , Arginina/química , Sequência Conservada , Reparo do DNA por Junção de Extremidades/fisiologia , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/fisiologia , Ensaio de Desvio de Mobilidade Eletroforética , Glicina/química , Humanos , Modelos Moleculares , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Oligodesoxirribonucleotídeos/metabolismo , Conformação Proteica , Domínios Proteicos , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The DNA-dependent protein kinase (DNA-PK) plays an essential role in nonhomologous DNA end joining (NHEJ) by initially recognizing and binding to DNA breaks. We have shown that in vitro, purified DNA-PK undergoes autophosphorylation, resulting in loss of activity and disassembly of the kinase complex. Thus, we have suggested that autophosphorylation of the DNA-PK catalytic subunit (DNA-PKcs) may be critical for subsequent steps in DNA repair. Recently, we defined seven autophosphorylation sites within DNA-PKcs. Six of these are tightly clustered within 38 residues of the 4,127-residue protein. Here, we show that while phosphorylation at any single site within the major cluster is not critical for DNA-PK's function in vivo, mutation of several sites abolishes the ability of DNA-PK to function in NHEJ. This is not due to general defects in DNA-PK activity, as studies of the mutant protein indicate that its kinase activity and ability to form a complex with DNA-bound Ku remain largely unchanged. However, analysis of rare coding joints and ends demonstrates that nucleolytic end processing is dramatically reduced in joints mediated by the mutant DNA-PKcs. We therefore suggest that autophosphorylation within the major cluster mediates a conformational change in the DNA-PK complex that is critical for DNA end processing. However, autophosphorylation at these sites may not be sufficient for kinase disassembly.