Analysis of the mechanism of interaction of simian Ku protein with DNA

Paillard, Sophie; Strauss, François

doi:10.1093/nar/19.20.5619

Cited by 203 publications

(167 citation statements)

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“…Ku is capable of binding to hairpin structures, single-stranded nicks, and gaps in an apparently sequence-independent manner (44)(45)(46)61). A number of studies have also suggested that the Ku protein is capable of sequence-specific DNA binding (36)(37)(38)(39)(40)60).…”

Section: Discussionmentioning

confidence: 99%

DNA looping by Ku and the DNA-dependent protein kinase

Cary

Peterson

Wang

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

228

138

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The DNA-dependent protein kinase (DNA-PK) is required for DNA double-strand break (DSB) repair and immunoglobulin gene rearrangement and may play a role in the regulation of transcription. The DNA-PK holoenzyme is composed of three polypeptide subunits: the DNA binding Ku70͞86 heterodimer and an Ϸ460-kDa catalytic subunit (DNA-PKcs). DNA-PK has been hypothesized to assemble at DNA DSBs and play structural as well as signal transduction roles in DSB repair. Recent advances in atomic force microscopy (AFM) have resulted in a technology capable of producing high resolution images of native protein and proteinnucleic acid complexes without staining or metal coating. The AFM provides a rapid and direct means of probing the protein-nucleic acid interactions responsible for DNA repair and genetic regulation. Here we have employed AFM as well as electron microscopy to visualize Ku and DNA-PK in association with DNA. A significant number of DNA molecules formed loops in the presence of Ku. DNA looping appeared to be sequence-independent and unaffected by the presence of DNA-PKcs. Gel filtration of Ku in the absence and the presence of DNA indicates that Ku does not form nonspecific aggregates. We conclude that, when bound to DNA, Ku is capable of self-association. These findings suggest that Ku binding at DNA DSBs will result in Ku self-association and a physical tethering of the broken DNA strands.

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Section: Discussionmentioning

confidence: 99%

DNA looping by Ku and the DNA-dependent protein kinase

Cary

Peterson

Wang

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

228

138

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“…In all of the above studies, the r R N A promoter was positioned close to the end of the linear D N A molecule. Although D N A ends are required for the initial binding of K u / D N A -P K to DNA, previous reports have suggested that once bound to the template, it can then translocate to internal positions on the D N A molecule (de Vries et al 1989;Paillard and Strauss 1991;Gottlieb and Jackson 19931. We were thus prompted to test w h e t h e r transcriptional i n h i b i t i o n was affected by the location of the D N A ends in regard to the transcription complex.…”

Section: Dna-pk and The Pol I Transcription Complex Must Be Colocalizmentioning

confidence: 99%

“…Ku, originally identified as an antigen recognized by sera from various autoimmune patients, is a moderately abundant nuclear protein comprising polypeptides of -70 and 80 kD in a tightly associated heterodimeric complex (Francoeur et al 1986;Mimori and Hardin 1986;Yaneva and Busch 1986;Reeves and Sthoeger 1989). Although some selective interactions with defined promoter elements have been described, most evidence indicates that Ku binds DNA in an essentially sequence-independent fashion (Mimori and Hardin 1986;de Vries et al 1989;Paillard and Strauss 1991;Zhang and Yaneva 1992;Blier et al 1993).…”

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confidence: 99%

DNA-dependent protein kinase: a potent inhibitor of transcription by RNA polymerase I.

et al. 1995

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DNA-dependent protein kinase (DNA-PK) comprises a catalytic subunit of -350 kD (p350) and a DNA-binding component termed Ku. Although DNA-PK can phosphorylate many transcription factors, no function for this enzyme in transcription has been reported thus far. Here, we show that DNA-PK strongly represses transcription by RNA polymerase I (Pol I). Transcriptional repression by DNA-PK requires ATP hydrolysis, and DNA-PK must be colocalized on the same DNA molecule as the Pol I transcription machinery. Consistent with DNA-PK requiring DNA ends for activity, transcriptional inhibition only occurs effectively on linearized templates. Mechanistic studies including single-round transcriptions, abortive initiation assays, and factor-independent transcription on a tailed template demonstrate that DNA-PK inhibits initiation (i.e., the formation of the first phosphodiester bonds) but does not affect transcription elongation. Repression of transcription involves phosphorylation of the transcription initiation complex, and rescue experiments reveal that the inactivated factor remains bound to the promoter and thus prevents initiation complex formation. We discuss the possible relevance of these findings in regard to the control of rRNA synthesis in vivo.

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“…Complex 1 is thought to result from binding of a single rKu to the dsDNA, while complex 2 is likely to contain 2 rKu per 48 bp duplex. Ku is thought to require a minimum of 20-25 bp for stable binding, and loading of multiple Ku molecules onto longer strands of duplex DNA has been observed [16]. It is worth noting that Figure 3A shows that at 7.7 nM Ku all of the 5 nM labeled DNA substrate has been bound, suggesting that a high percentage of the rKu is DNA-binding active.…”

mentioning

confidence: 96%

2-Step purification of the Ku DNA repair protein expressed in Escherichia coli

Hanakahi

2007

Protein Expression and Purification

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The Ku protein is involved in DNA double-strand break repair by non-homologous end-joining (NHEJ), which is crucial to the maintenance of genomic integrity in mammals. To study the role of Ku in NHEJ we developed a bicistronic E. coli expression system for the Ku70 and Ku80 subunits. Association of the Ku70 and Ku80 subunits buries a substantial amount of surface area (~9000Å 2 [1]), which suggests that herterodimerization may be important for protein stability. N-terminally his 6 -tagged Ku80 was soluble in the presence, but not in the absence, of bicistronically expressed untagged Ku70. In a 2-step purification, metal chelating affinity chromatography was followed by step-gradient elution from heparin-agarose. Co-purification of equimolar amounts of his 6 -tagged Ku80 and untagged Ku70 was observed, which indicated heterodimerization. Recombinant Ku bound dsDNA, activated the catalytic subunit of the DNA-dependent kinase (DNA-PKcs) and functioned in NHEJ reactions in vitro. Our results demonstrate that while the heterodimeric interface of Ku is extensive it is nonetheless possible to produce biologically active Ku protein in E. coli. KeywordsKu; DNA-PK; non-homologous end-joining; NHEJ The repair of DNA double strand breaks (DSB) is critical to the maintenance of genomic integrity. The importance of DSB repair is highlighted by the fact that unrepaired DSBs can result in the loss of genetic information, chromosomal translocation and even cell death [2]. An important mechanism for DSB repair is non-homologous end-joining (NHEJ) -a homology independent process that is used for the repair DSBs during G0, G1 and early-S phases of the cell cycle [2]. As most of the cells in the mammalian body are terminally differentiated (G0), NHEJ is the major DSB repair pathway used by mammals.Thus far, six mammalian NHEJ proteins have been described. Central to the DSB repair process is DNA Ligase IV and the proteins XRCC4 and XLF, which are though to function as a complex in the ligation reaction [3][4][5][6] (Fig. 1C). Three other proteins -the Ku heterodimer ( Fig. 1) and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) -comprise the DNAdependent protein kinase (DNA-PK) [7]. Studies employing mouse models have demonstrated that genomic stability depends upon the fidelity of DSB repair through the NHEJ pathway and deficiencies in NHEJ have been linked to cancer [2,8].Correspondence to: Les A. Hanakahi. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. The DNA end-binding protein Ku protein plays an important role in mammalian NHEJ. Owing to its ring-shaped structure,...

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Analysis of the mechanism of interaction of simian Ku protein with DNA

Cited by 203 publications

References 22 publications

DNA looping by Ku and the DNA-dependent protein kinase

DNA looping by Ku and the DNA-dependent protein kinase

DNA-dependent protein kinase: a potent inhibitor of transcription by RNA polymerase I.

2-Step purification of the Ku DNA repair protein expressed in Escherichia coli

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