FIG. 4.

EMSA of mutant Rep78 synthesized in vitro. Three microliters of in vitro-synthesized Rep78 or mutant Rep protein in the absence of radiolabeled amino acids was incubated for 15 min at 30°C with 20,000 cpm of 5′-end-labeled RBS oligonucleotide probe in a 10-μl solution of 10 mM HEPES-KOH (pH 7.9), 50 mM KCl, 0.1 mM EDTA, 0.05% BSA, 10% glycerol, and 1 μg of sheared calf thymus DNA. Reaction products were separated on 4% nondenaturing polyacrylamide gels, dried, and then analyzed on a BAS-1500 imaging analyzer. Unprogrammed lysate (no DNA) and lysate programmed with vector (vector) were also included as negative controls. ∗, complete loss of binding activity; ∗∗, consistently lower binding activity compared to the wild type.