Purification of purine-box regulator complexes reveals ARRE–DNA-binding subunits NF90, NF45, Ku80 and Ku70

Abstract

<p><b>Copyright information:</b></p><p>Taken from "Dynamic binding of Ku80, Ku70 and NF90 to the IL-2 promoter in activated T-cells"</p><p></p><p>Nucleic Acids Research 2007;35(7):2302-2310.</p><p>Published online 27 Mar 2007</p><p>PMCID:PMC1874627.</p><p>© 2007 The Author(s)</p> EMSA were used to monitor enrichment of ARRE–DNA-binding complexes from nuclear extracts of Jurkat T-cells stimulated with PMA + Ionomycin. () Diethylaminoethyl (DEAE) elution. Fractions enriched in complex I (and small amounts of complexes II and III) were pooled and dialyzed. () Carboxymethyl (CM) elution. Note the substantial conversion of complex I into complexes II and III. Fractions containing complex I only were pooled and dialyzed. () Octylamine elutions with steps of KCl. Right, oligonucleotide competitions demonstrate greater inhibition of complex I in elution 0.2 with 50 ng of wild type (W) compared to mutant (M) ARRE, yet 50 ng of M does produce substantial inhibition of complex I. () DNA-affinity elution. Octylamine elutions 0.2 M KCl/pool A, enriched in complex I only, were diluted and loaded onto a mutant ARRE–DNA-affinity column and eluted with the indicated steps of KCl. Note the presence of complexes I and III in eluted fraction 0.2, associated with principal protein subunits at 45, 80, 90, 130 and 350 kDa shown by SDS–PAGE with silver staining. Ten nanograms of wild-type (W) ARRE oligonucleotide inhibits complex I and promotes conversion to complex III more potently than mutant (M) oligonucleotide. () DNA-affinity elution. Octylamine elutions enriched in complexes II and III, pool B, were diluted and loaded onto a wild-type ARRE–DNA-affinity column, and maximal DNA-binding activity was eluted at 0.4 M KCl, associated with protein subunits at 70, 80 and 350 kDa shown by SDS–PAGE with silver staining. Internal tryptic peptide sequences of the 70 and 80 kDa proteins identified them as Ku70 and Ku80. Western immunoblot with human JM anti-Ku sera (right) confirms the identity of Ku70 and Ku80 (lane 4), and suggests the slowest migrating protein to be DNA-dependent protein kinase (DPK) catalytic subunit