Detection and isolation of minisatellite Pc-1 binding proteins

Minisatellites (MNs) are arrays of 5-100 nucleotide repeats that are disper sed throughout the genome of vertebrates. They demonstrate alteration in tu mors and in cells exposed to various carcinogens, but the molecular mechani sms underlying the induction of mutations at MNs are largely unknown. Hyper variable MN Pc-1 isolated from the mouse genome consists of tandem repeats of d(GGCAG) flanked with locus-specific sequences at both ends. We have fou nd that MN mutations are induced in NIH3T3 cells by treatment with okadaic acid using a Pc-1 MN fragment as a probe. In order to shed light on the mol ecular mechanisms, we isolated six MN Pc-1 binding proteins, pA, pB pD, pE, pF and pG, from nuclear extracts of NIH3T3 cells treated with okadaic acid . While pA and pB bound to the G-rich strand of Pc-1, pD, pE, pF and pG bou nd to the complementary C-rich strand. Sequence specificities for DNA bindi ng were revealed and one base substitution and insertion into the Pc-l repe at unit dramatically changed the affinity of each protein, suggesting that they bind to Pc-1 and Pc-1-like MNs in vivo. (C) 2001 Elsevier Science B.V. All rights reserved.