Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair

Walker, John R.; Corpina, Richard A.; Goldberg, Jonathan M.

doi:10.1038/35088000

Cited by 1,044 publications

(1,144 citation statements)

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“…Structurally, Ku is unusual in that heterodimerization of the Ku70 and Ku80 subunits buries a large amount of surface area (~9000Å 2 [1]). Perhaps as a result of the extensive interfacial surface, we found that soluble Ku could not be produced by independently expressing the two subunits.…”

Section: Resultsmentioning

confidence: 99%

“…These data indicate that E. coli produced rKu is suitable for use in the biochemical analysis of mammalian NHEJ. Space-filling model of Ku showing Ku70 (red) and Ku80 (gold) generated using Protein Databank accession number 1JEY [1]. A) Side-view.…”

Section: Resultsmentioning

confidence: 99%

“…A) Side-view. Ring-structure of Ku capable of encircling 2 turns of dsDNA, which passes thru the opening of the preformed ring [1]. B) Topview.…”

Section: Resultsmentioning

confidence: 99%

“…To study the role of Ku in NHEJ we have established an E. coli expression system for Ku, which will eliminate the need for eukaryotic cell culture. Heterodimerization of the Ku subunits buries 9000 Å 2 [1] (Fig. 1); an unusually large amount when compared with most other 2 subunit multimerization events that typically bury <5000 Å 2 [10].…”

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confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…A) Side-view. Ring-structure of Ku capable of encircling 2 turns of dsDNA, which passes thru the opening of the preformed ring [1]. B) Topview.…”

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Hanakahi

2007

Protein Expression and Purification

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“…The structure of the Ku70/80 heterodimer directly shows why this protein binds specifically to DNA ends: its ring shape allows it to thread onto a double-stranded DNA molecule, but not to internal DNA positions (Walker et al, 2001). As expected on the basis of this structure, the protein can slide along a DNA molecule to internal positions without the need for an energy source, such as ATP (de Vries et al, 1989;Ristic et al, 2003).…”

Section: Dna-pkmentioning

confidence: 91%

Non-homologous end-joining, a sticky affair

2007

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Rejoining of broken chromosomes is crucial for cell survival and prevention of malignant transformation. Most mammalian cells rely primarily on the non-homologous end-joining pathway of DNA double-strand break (DSB) repair to accomplish this task. This review focuses both on the core non-homologous end-joining machinery, which consists of DNA-dependent protein kinase and the ligase IV/XRCC4 complex, and on accessory factors that facilitate rejoining of a subset of the DSBs. We discuss how the ATM protein kinase and the Mre11/Rad50/Nbs1 complex might function in DSB repair and what role ionizing radiation-induced foci may play in this process.

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Lymphoid Malignancies

Teitell

Pandolfi

2004

Mouse Models of Human Cancer

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Malignant transformation may occur at any point during lymphoid cell maturation, resulting in stage‐ and lineage‐specific cancers of the immune system. Many in‐bred and engineered mouse strains develop specific lymphoid cancers, providing potentially useful models of human malignancy with insight for pathogenetic mechanisms. Newer techniques, targeting dysregulated gene expression to specific developmental stages portend newer, more accurate mouse models in the future. These models will likely have an increasing role in evaluating targeted therapeutic strategies for efficacy.

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Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair

Cited by 1,044 publications

References 44 publications

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

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

Non-homologous end-joining, a sticky affair

Lymphoid Malignancies

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