cDNA-derived Amino Acid Sequence of the 86-kDa Subunit of the Ku Antigen

Yaneva, Mariana; Wen, Juan; Ayala, Alma Leticia Martínez; Cook, R G

doi:10.1016/s0021-9258(18)80011-4

Cited by 118 publications

(10 citation statements)

References 24 publications

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“…Although the genes encoding the Ku70 and Ku80 subunits of DNA-PK had been cloned at this stage (Reeves and Sthoeger, 1989;Yaneva et al, 1989), the PRKDC gene encoding DNA-PKcs had not. A breakthrough came in early 1995, when the DNA-PKcs cDNA was cloned and found to encode a colossal 460-kDa polypeptide (Hartley et al, 1995).…”

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confidence: 99%

ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response

2017

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In vertebrate cells, the DNA damage response is controlled by three related kinases: ATM, ATR, and DNA-PK. It has been 20 years since the cloning of ATR, the last of the three to be identified. During this time, our understanding of how these kinases regulate DNA repair and associated events has grown profoundly, although major questions remain unanswered. Here, we provide a historical perspective of their discovery and discuss their established functions in sensing and responding to genotoxic stress. We also highlight what is known regarding their structural similarities and common mechanisms of regulation, as well as emerging non-canonical roles and how our knowledge of ATM, ATR, and DNA-PK is being translated to benefit human health.

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confidence: 99%

ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response

2017

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“…X represents unidentifiable residues during the sequence call. The deduced amino acid sequences for the 80-kDa subunit (Mimori et al, 1989;Yaneva et al, 1989) and 70-kDa (Reeves & Sthoeger, 1989) subunits of human Ku protein were from published results. FPLC, Figure 1) using monoclonal antibodies to the 80-kDa (antibody P6D8) and 70-kDa (antibody N3H10) subunits of human Ku proteins (Porges et al, 1990) showed the presence of two protein bands of about 83 and 70 kDa.…”

Section: Resultsmentioning

confidence: 99%

“…The DNA-ATPase activity that is intrinsic to the Ku-like protein reported here requires single-stranded DNA as a cofactor. The activation of the ATPase activity Koonin, 1993 Mimori & Hardin, 1986; Yaneva et al, 1989 Reeves & Sthoeger, 1989 0 X signifies any residue. U signifies a hydrophobic residue.…”

Section: Discussionmentioning

confidence: 99%

“…The human Ku autoantigen is a heterodimeric protein composed of equimolar amounts of 80and 70-kDa subunits (Mimori et al, 1981(Mimori et al, ,1986Reeves, 1985;Yaneva etal., 1985). Full-length cDNA clones have been obtained for both subunits of the human (Reeves & Sthoeger, 1989;Yaneva et al,, 1989;Mimori et al, 1990) and murine (Chan et al, 1989;Porges et al, 1990;Falzon & Kuff, 1992) Ku proteins. The actual molecular masses for the deduced primary sequences of the respective subunits are 83 kDa (Mimori et al, 1990) and 70 kDa (Reeves & Sthoeger, 1989).…”

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confidence: 99%

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DNA-Dependent ATPase from HeLa Cells Is Related to Human Ku Autoantigen

Cao¹,

Pitt²,

Leszyk³

et al. 1994

Biochemistry

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A 150-kDa DNA-dependent ATPase composed of 83/68-kDa subunits was previously reported to cofractionate with a 21S complex of enzymes for DNA synthesis from HeLa cells (Vishwanatha, J. K., & Baril, E. F. (1990) Biochemistry 29, 8753-8759). The DNA-dependent ATPase was purified to electrophoretic homogeneity from a HeLa cell homogenate by a modified procedure that involves subcellular fractionation, poly(ethylene-glycol) precipitation of the combined nuclear extract/cytosol, and chromatography on Q-Sepharose and native and denatured DNA/celluloses followed by Mono-S fast protein liquid chromatography. The purified enzyme showed equimolar amounts of 83- and 68-kDa polypeptides following polyacrylamide gel electrophoresis under denaturing conditions. Sequence analysis of peptide fragments derived from the separated 83- and 68-kDa polypeptides showed 90-100% homology with the corresponding 80- and 70-kDa subunits of human Ku protein. Immunoblot analysis of the ATPase during the course of its purification and immunoprecipitation with antibodies to the 80- and 70-kDa subunits of human Ku protein confirmed the relationship of the 83- and 68-kDa polypeptides of the human DNA-dependent ATPase to the subunits of human Ku protein. Both the 83- and 68-kDa polypeptides are phosphorylated by a DNA-dependent protein kinase that cofractionates with the ATPase. The DNA-dependent ATPase activity is up regulated by phosphorylation.

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“…However, it remains to be determined if dimerization occurs in the cytoplasm or the nucleus, and whether chaperonins such as hsc-70 (Shi and Thomas, 1992) are involved in the nuclear transport of p70 and p80. Although both p70 (Reeves and Sthoeger, 1989) and p80 (Yaneva et al, 1989) contain sequences resembling nuclear localization motifs, further studies will be necessary to define their respective roles in facilitating nuclear transport.…”

Section: Nuclear Transport and Assembly Of P70 And P80mentioning

confidence: 99%

Assembly and DNA binding of recombinant Ku (p70/p80) autoantigen defined by a novel monoclonal antibody specific for p70/p80 heterodimers

Wang

Satoh

Pierani

et al. 1994

Journal of Cell Science

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The Ku autoantigen is a heterodimer of 70 kDa (p70) and -80 kDa (p80) subunits that is the DNA-binding component of a DNA-dependent protein kinase (DNA-PK). The 350 kDa (p350) catalytic subunit of DNA-PK phosphorylates Sp-1, Oct-1, p53 and RNA polymerase II in vitro, but the precise cellular role of DNA-PK remains unclear. In the present studies, the assembly of p70/p80 heterodimers and the interaction of Ku with DNA was investigated using recombinant vaccinia viruses directing the synthesis of human p70 (p70-vacc) and p80 (p80-vacc), and monoclonal antibodies (mAbs). Expression of human Ku antigens in rabbit kidney (RK13) cells could be demonstrated by immunofluorescent staining because this cell line contains little endogenous Ku. A novel mAb designated 162 stained the nuclei of RK13 cells coinfected with p70-vacc and p80-vacc, but not cells that were infected with either virus alone, suggesting that it recognized the p70/p80 heterodimer but not monomeric p70 or p80. In agreement with the immunofluorescence data, 162 immunoprecipitated both p70 and p80 from extracts of coinfected cells, but did not immunoprecipitate either subunit by itself from extracts of cells infected with p70-vacc or p80-vacc, respectively. Conversely, the binding of 162 to Ku isolated from human K562 cells stabilized the p70/p80 heterodimer under conditions that normally dissociate p70 from p80. The nuclei of cells infected with p70-vacc alone could be stained with mAb N3H10 (anti-p70) and cells infected with p80-vacc alone could be stained with mAb 111 (anti-p80), indicating that the formation of p70/p80 heterodimers was not required for nuclear transport. Finally, free recombinant and cellular p70 both bound to DNA efficiently in vitro, suggesting that free p70, like the p70/p80 heterodimer, serves as a DNA-binding factor. Moreover, free human p70 could be released from the nuclei of p70-vacc-infected RK13 cells by deoxyribonuclease I treatment, suggesting that it was associated with chromatin in vivo. The nuclear transport of free p70 and the association of free p70 with chromatin in vivo raise the possibility that newly synthesized cellular p70 might undergo nuclear transport and DNA-binding prior to dimerization with p80 or assembly with p350.

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cDNA-derived Amino Acid Sequence of the 86-kDa Subunit of the Ku Antigen

Cited by 118 publications

References 24 publications

ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response

ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response

DNA-Dependent ATPase from HeLa Cells Is Related to Human Ku Autoantigen

Assembly and DNA binding of recombinant Ku (p70/p80) autoantigen defined by a novel monoclonal antibody specific for p70/p80 heterodimers

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