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Título: | Implication of DNA Polymerase λ in Alignment-based Gap Filling for Nonhomologous DNA End Joining in Human Nuclear Extracts |
Autor: | Wan Lee, Jae; Blanco, Luis CSIC ORCID ; Zhou, Tong; García-Díaz, Miguel; Bebenek, Katarzyna; Kunkel, Thomas A.; Wang, Zhigang; Povirk, Lawrence F. | Fecha de publicación: | 2-ene-2004 | Editor: | Elsevier American Society for Biochemistry and Molecular Biology |
Citación: | Journal of Biological Chemistry 279(1): 805-811 (2004) | Resumen: | Accurate repair of free radical-mediated DNA double-strand breaks by the nonhomologous end joining pathway requires replacement of fragmented nucleotides in the aligned ends by a gap-filling DNA polymerase. Nuclear extracts of human HeLa cells, supplemented with recombinant XRCC4-DNA ligase IV complex (XRCC4/ligase IV), were capable of accurately rejoining model double-strand break substrates with a 1- or 2-base gap, and the gap-filling step was dependent on XRCC4/ligase IV. To determine what polymerase was responsible for gap filling, end joining was examined in the presence of polyclonal antibodies against each of two prime candidate enzymes, DNA polymerases μ and λ, both of which were present in the extracts. For a DNA substrate with partially complementary 3′ overhangs and a 2-base gap, antibodies to polymerase λ completely eliminated both gap filling and accurate end joining, whereas antibodies to polymerase μ had little effect. Immunodepletion of polymerase λ, but not polymerase μ, likewise blocked both gap filling and end joining, and both functions could be restored by addition of recombinant polymerase λ. Recombinant polymerase μ, and a truncated polymerase λ lacking the Brca1 C-terminal domain, were at least 10-fold less active in restoring gap filling to the immunodepleted extracts, and polymerase β was completely inactive. The results suggest that polymerase λ is the primary gap-filling polymerase for accurate nonhomologous end joining, and that the Brca1 C-terminal domain is required for this activity. | Versión del editor: | http://dx.doi.org/10.1074/jbc.M307913200 | URI: | http://hdl.handle.net/10261/338875 | DOI: | 10.1074/jbc.M307913200 | Identificadores: | issn: 0021-9258 e-issn: 1083-351X |
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