Search

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. [Display omitted] ● PolλKGET can prime synthesis from an unpaired DSB end, previously unique to Polμ. ● PolλKGET synthesis from unpaired ends also 'skips ahead', like Polμ. ● PolλKGET retains ability to prime synthesis from paired ends, like wildtype Polλ. ● Crystal structure of PolλKGET engaged in synapsis with unpaired DSB ends. ● PolλKGET Loop1 sequence is unexpectedly essential for its unique activity. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Highlights: [•] Cdk2/cyclin A complex phosphorylates human Polμ in vitro. [•] Ser12, Thr21 (BRCT domain) and Ser372 (loop1) are the Cdk target residues. [•] Phospho-mimicking S372E impaired terminal transferase and end-joining activities. [•] Polμ gap-filling activity was not affected by S372E mutation. [•] Polμ error-prone activities might be cell cycle regulated by phosphorylation. [ABSTRACT FROM AUTHOR]