RESUMEN
DNA polymerases lambda (Polλ) and mu (Polµ) are X-Family polymerases that participate in DNA double-strand break (DSB) repair by the nonhomologous end-joining pathway (NHEJ). Both polymerases direct synthesis from one DSB end, using template derived from a second DSB end. In this way, they promote the NHEJ ligation step and minimize the sequence loss normally associated with this pathway. The two polymerases differ in cognate substrate, as Polλ is preferred when synthesis must be primed from a base-paired DSB end, while Polµ is required when synthesis must be primed from an unpaired DSB end. We generated a Polλ variant (PolλKGET) that retained canonical Polλ activity on a paired end-albeit with reduced incorporation fidelity. We recently discovered that the variant had unexpectedly acquired the activity previously unique to Polµ-synthesis from an unpaired primer terminus. Though the sidechains of the Loop1 region make no contact with the DNA substrate, PolλKGET Loop1 amino acid sequence is surprisingly essential for its unique activity during NHEJ. Taken together, these results underscore that the Loop1 region plays distinct roles in different Family X polymerases.
Asunto(s)
ADN Polimerasa beta , ADN Polimerasa Dirigida por ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Mutación con Ganancia de Función , ADN Polimerasa beta/metabolismo , Reparación del ADN , ADN/metabolismo , Reparación del ADN por Unión de ExtremidadesRESUMEN
Non-homologous end joining (NHEJ) is the main mechanism for double strand break (DSB) DNA repair. The error-prone DNA polymerase mu (Polµ) is involved in immunoglobulin variable region rearrangement and in general, NHEJ in non-lymphoid cells. Deletion of NHEJ factors in P53-/- mice, which are highly prone to development of T cell lymphoma, generally increases cancer incidence and shifts the tumor spectrum towards aggressive pro-B lymphoma. In contrast, Polµ deletion increased sarcoma incidence, proportionally reducing pro-B lymphoma development on the P53-deficient background. Array comparative genomic hybridization (aCGH) analyses showed DNA copy number alterations in both P53-/- and Polµ-/-P53-/- lymphomas. Our results also indicate that the increase in sarcoma incidence in Polµ-/-P53-/- mice could be associated with Cdk4 and Kub3 amplification and overexpression. These results identify a role for Polµ in the prevention of sarcomagenesis on a murine P53-deficient background, in contrast to most other NHEJ factors.
Asunto(s)
Carcinogénesis , Reparación del ADN por Unión de Extremidades , ADN Polimerasa Dirigida por ADN/genética , Sarcoma/metabolismo , Proteína p53 Supresora de Tumor/genética , Animales , Proteínas Portadoras/genética , Quinasa 4 Dependiente de la Ciclina/genética , ADN/metabolismo , Variaciones en el Número de Copia de ADN , Amplificación de Genes , Eliminación de Gen , Inestabilidad Genómica , Linfoma/genética , Linfoma/metabolismo , Linfoma/patología , Ratones , Ratones Noqueados , Sarcoma/genética , Sarcoma/patología , Regulación hacia ArribaRESUMEN
Nonhomologous end joining (NHEJ) repairs chromosome breaks and must remain effective in the face of extensive diversity in broken end structures. We show here that this flexibility is often reliant on the ability to direct DNA synthesis across strand breaks, and that polymerase (Pol) µ and Pol λ are the only mammalian DNA polymerases that have this activity. By systematically varying substrate in cells, we show each polymerase is uniquely proficient in different contexts. The templating nucleotide is also selected differently, with Pol µ using the unpaired base adjacent to the downstream 5' phosphate even when there are available template sites further upstream of this position; this makes Pol µ more flexible but also less accurate than Pol λ. Loss of either polymerase alone consequently has clear and distinguishable effects on the fidelity of repair, but end remodeling by cellular nucleases and the remaining polymerase helps mitigate the effects on overall repair efficiency. Accordingly, when cells are deficient in both polymerases there is synergistic impact on NHEJ efficiency, both in terms of repair of defined substrates and cellular resistance to ionizing radiation. Pol µ and Pol λ thus provide distinct solutions to a problem for DNA synthesis that is unique to this pathway and play a key role in conferring on NHEJ the flexibility required for accurate and efficient repair.
Asunto(s)
Reparación del ADN por Unión de Extremidades , ADN Polimerasa beta/química , ADN Polimerasa Dirigida por ADN/química , Animales , Proliferación Celular , ADN/química , Daño del ADN , Relación Dosis-Respuesta en la Radiación , Fibroblastos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nucleótidos/química , Radiación IonizanteRESUMEN
Among the many proteins used to repair DNA double-strand breaks by nonhomologous end joining (NHEJ) are two related family X DNA polymerases, Pol λ and Pol µ. Which of these two polymerases is preferentially used for filling DNA gaps during NHEJ partly depends on sequence complementarity at the break, with Pol λ and Pol µ repairing complementary and noncomplementary ends, respectively. To better understand these substrate preferences, we present crystal structures of Pol µ on a 2-nt gapped DNA substrate, representing three steps of the catalytic cycle. In striking contrast to Pol λ, Pol µ "skips" the first available template nucleotide, instead using the template base at the 5' end of the gap to direct nucleotide binding and incorporation. This remarkable divergence from canonical 3'-end gap filling is consistent with data on end-joining substrate specificity in cells, and provides insights into polymerase substrate choices during NHEJ.
Asunto(s)
Reparación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/biosíntesis , Catálisis , Cristalografía por Rayos X , Daño del ADN , ADN Polimerasa beta/química , Humanos , Cinética , Conformación de Ácido Nucleico , Nucleótidos/genética , Estructura Secundaria de Proteína , Análisis de Secuencia de ADN , Especificidad por SustratoRESUMEN
Human family X polymerases contribute both to genomic stability and variability through their specialized functions in DNA repair. Polµ participates in the repair of spontaneous double strand breaks (DSB) by non homologous end-joining (NHEJ), and also in the V(D)J recombination process after programmed DSBs. Polµ plays this dual role due to its template-dependent and terminal transferase (template-independent) polymerization activities. In this study we evaluated if Polµ could be regulated by Cdk phosphorylation along the cell cycle. In vitro kinase assays showed that the S phase-associated Cdk2/cyclin A complex was able to phosphorylate Polµ. We identified Ser12, Thr21 (located in the BRCT domain) and Ser372 (located in loop1) as the target residues. Mutation of these residues to alanine indicated that Ser372 is the main phosphorylation site. Mobilization of loop1, which mediates DNA end micro-synapsis, is crucial both for terminal transferase and NHEJ. Interestingly, the phospho-mimicking S372E mutation specifically impaired these activities. Our evidences suggest that Polµ could be regulated in vivo by phosphorylation of the BRCT domain (Ser12/Thr21) and of Ser372, affecting the function of loop1. Consequently, Polµ's most distinctive activities would be turned off at specific cell-cycle phases (S and G2), when these promiscuous functions might be harmful to the cell.