DNA double-strand break repair in cell-free extracts from Ku80-deficient cells: implications for Ku serving as an alignment factor in non-homologous DNA end joining

Non-homologous DNA end joining (NHEJ) is considered the major pathway of double-strand break (DSB) repair in mammalian cells and depends, among other things, on the DNA end-binding Ku70/80 hetero-dimer. To investigate the function of Ku in NHEJ we have compared the ability of cell-free extracts from...

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Veröffentlicht in:Nucleic acids research 2000-07, Vol.28 (13), p.2585-2596
Hauptverfasser: Feldmann, E, Schmiemann, V, Goedecke, W, Reichenberger, S, Pfeiffer, P
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container_issue 13
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container_title Nucleic acids research
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creator Feldmann, E
Schmiemann, V
Goedecke, W
Reichenberger, S
Pfeiffer, P
description Non-homologous DNA end joining (NHEJ) is considered the major pathway of double-strand break (DSB) repair in mammalian cells and depends, among other things, on the DNA end-binding Ku70/80 hetero-dimer. To investigate the function of Ku in NHEJ we have compared the ability of cell-free extracts from wild-type CHO-K1 cells, Ku80-deficient xrs6 cells and Ku80-cDNA-complemented xrs6 cells (xrs6-Ku80) to rejoin different types of DSB in vitro. While the two Ku80-proficient extracts were highly efficient and accurate in rejoining all types of DNA ends, the xrs6 extract displayed strongly decreased NHEJ efficiency and accuracy. The lack of accuracy is most evident in non-homologous terminus configurations containing 3'-overhangs that abut a 5'-overhang or blunt end. While the sequences of the 3'-overhangs are mostly preserved by fill-in DNA synthesis in the Ku80-proficient extracts, they are always completely lost in the xrs6 extract so that, instead, small deletions displaying microhomology patches at their breakpoints arise. In summary, our results are consistent with previous results from Ku-deficient yeast strains and indicate that Ku may serve as an alignment factor that not only increases NHEJ efficiency but also accuracy. Furthermore, a secondary NHEJ activity is present in the absence of Ku which is error-prone and possibly assisted by base pairing interactions.
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To investigate the function of Ku in NHEJ we have compared the ability of cell-free extracts from wild-type CHO-K1 cells, Ku80-deficient xrs6 cells and Ku80-cDNA-complemented xrs6 cells (xrs6-Ku80) to rejoin different types of DSB in vitro. While the two Ku80-proficient extracts were highly efficient and accurate in rejoining all types of DNA ends, the xrs6 extract displayed strongly decreased NHEJ efficiency and accuracy. The lack of accuracy is most evident in non-homologous terminus configurations containing 3'-overhangs that abut a 5'-overhang or blunt end. While the sequences of the 3'-overhangs are mostly preserved by fill-in DNA synthesis in the Ku80-proficient extracts, they are always completely lost in the xrs6 extract so that, instead, small deletions displaying microhomology patches at their breakpoints arise. 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To investigate the function of Ku in NHEJ we have compared the ability of cell-free extracts from wild-type CHO-K1 cells, Ku80-deficient xrs6 cells and Ku80-cDNA-complemented xrs6 cells (xrs6-Ku80) to rejoin different types of DSB in vitro. While the two Ku80-proficient extracts were highly efficient and accurate in rejoining all types of DNA ends, the xrs6 extract displayed strongly decreased NHEJ efficiency and accuracy. The lack of accuracy is most evident in non-homologous terminus configurations containing 3'-overhangs that abut a 5'-overhang or blunt end. While the sequences of the 3'-overhangs are mostly preserved by fill-in DNA synthesis in the Ku80-proficient extracts, they are always completely lost in the xrs6 extract so that, instead, small deletions displaying microhomology patches at their breakpoints arise. In summary, our results are consistent with previous results from Ku-deficient yeast strains and indicate that Ku may serve as an alignment factor that not only increases NHEJ efficiency but also accuracy. Furthermore, a secondary NHEJ activity is present in the absence of Ku which is error-prone and possibly assisted by base pairing interactions.</abstract><cop>England</cop><pub>Oxford Publishing Limited (England)</pub><pmid>10871410</pmid><doi>10.1093/nar/28.13.2585</doi><tpages>12</tpages><oa>free_for_read</oa></addata></record>
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subjects Animals
Antigens, Nuclear
Base Pairing
Base Sequence
Cell Extracts
CHO Cells
Cricetinae
Dimerization
DNA - chemistry
DNA - genetics
DNA - metabolism
DNA Helicases
DNA Repair - genetics
DNA-Binding Proteins - deficiency
DNA-Binding Proteins - genetics
DNA-Binding Proteins - metabolism
Genetic Complementation Test
Ku Autoantigen
Ku70 antigen
Ku80 antigen
Models, Genetic
Nuclear Proteins - deficiency
Nuclear Proteins - genetics
Nuclear Proteins - metabolism
Recombination, Genetic - genetics
Saccharomyces cerevisiae Proteins
Sequence Analysis, DNA
Sequence Homology, Nucleic Acid
Substrate Specificity
Thermodynamics
title DNA double-strand break repair in cell-free extracts from Ku80-deficient cells: implications for Ku serving as an alignment factor in non-homologous DNA end joining
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