Figure 3

Characterization of the DAPB binding domain in the 5'- APP -UTR. (A) DNase I footprinting of a wild type APP promoter fragment extending from position -40 including the UTR to position +147 (lanes 1 and 6). The coding strand was 5' end-labeled with [³²P] and the resulting fragment was digested with DNase I either in the presence (+) or absence (-) of nuclear extract. Brackets delineate the position of DNase I protected domain DAPB from position +72 to +115 as discussed in the text. (B) Mobility shift electrophoresis with elution fractions from SP ion exchange chromatography of whole HeLa cell nuclear extract and a [³²P] 5' end-labeled double stranded oligonucleotide containing the 5'-APP-UTR sequence from position +60 to +120 [60-120]. The binding complex eluted predominantly in fractions 11-13 (lanes 3-5). The binding complex (b) and the free oligonucleotide (f) are indicated by brackets throughout the figure. (C) Mobility shift competition with the 5'-APP-UTR fragment [60-120] as a labeled probe: Mobility shift without competitor (lane 1); mobility shift with a 5-fold (lane 2) and 20-fold (lane 3) molar excess of unlabeled wild type [60-120] sequence; competition with 5-fold (lane 4), 20-fold (lane 5), and 100-fold (lane 6) excess of unlabeled oligonucleotide containing transverse mutations from position +86 to +100 (M [86-100]) within the [60-120] fragment (lower panel); competition with a 5-fold (lane 7), 20-fold (lane 8), and 100-fold (lane 9) excess of unlabeled oligonucleotide containing transverse mutations from position +71 to +85 (M [81-85]). (D) Mobility shift competition with the [60-120] fragment as a labeled probe (lane 1). Self-competition with a 10-fold (lane 2) and 40-fold (lane 3) excess of wild type [60-120] sequence and competition with a 40-fold unlabeled excess of mutations M [86-90] (lane 4), M [91-95] (lane 5), M [96-100] (lane 6), M [101-105] (lane 7), and M [86-100] (lane 8).