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

Abstract

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,...

A Human Short Open Reading Frame (sORF)-encoded Polypeptide That Stimulates DNA End Joining

“…Finally, we measured the effect of purified MRI-2 on NHEJ efficiency in vitro using a double-stranded DNA ligation assay in cell extracts (18,22,37). Purified recombinant MRI-2 was added to NHEJ-competent cell extracts, and the conversion of a radiolabeled DNA duplex to higher molecular weight oligomeric ligation products was monitored.…”

“…Protein expression was induced with 1 mM isopropyl-1-thio-␤-D-galactopyranoside, and cells were grown at 30°C with shaking for 4 h. Cells were harvested by centrifugation at 8000 rpm for 10 min at 4°C and stored at Ϫ80°C until use. Protein was purified using a protocol previously reported for Ku (18). Pure MRI-2 (assayed by SDS-PAGE) was dialyzed into 50 mM Tris-HCl, pH 8.0, 20% glycerol, 50 mM NaCl, and 1 mM DTT, aliquoted, and flashfrozen.…”

PAXX Is an Accessory c-NHEJ Factor that Associates with Ku70 and Has Overlapping Functions with XLF

“…Full-length Ku70, C-terminally tagged with six histidines, was produced in bacteria using a construct pMM1271 obtained by cloning the Ku70 cDNA with a C-terminal poly-histidine tag in between the NdeI and XhoI sites of pETduet-1 (Novagen). Ku70 purification from bacteria was performed as described (Hanakahi, 2007).…”

SFPQ•NONO and XLF function separately and together to promote DNA double-strand break repair via canonical nonhomologous end joining

“…Repair factors were expressed Escherichia coli and purified by immobilized metal affinity chromatography, followed in most cases by Superdex S200 gel filtration. Procedures for expression and purification of factors other than SFPQ•NONO were based on modifications of existing protocols ( 38 – 41 ). Details are provided in Supplementary Data .…”