High-affinity binding of the cell cycle-regulated transcription factors E2F1 and E2F4 to benzo(a)pyrene diol epoxide-DNA adducts.

摘要

Previous studies indicated that DNA adducts formed by a carcinogenic diol epoxide, 7r,8t-dihydroxy-9t, 10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), can increase the affinity of the transcription factor Sp1 for DNA sequences that are not normally specific binding sites. It was suggested that adduct-induced bends in the DNA were responsible for this behavior. The cell cycle-regulated transcription factor E2F is also known to bend DNA upon binding. When partially purified E2F was tested in a gel mobility-shift assay, binding to a target DNA containing two consensus E2F-binding sites was enhanced by prior modification of the DNA with BPDE. Recombinant human E2F1, E2F4, and DP1 fusion proteins were affinity purified from bacteria expressing these genes. A combination of either E2F1 or E2F4 with their dimerization partner, DP1, gave preparations that exhibited binding to the E2F site-containing DNA fragment. In both cases, the proteins exhibited much higher apparent affinity for BPDE-modified DNA thanfor unmodified DNA. In addition, BPDE-modified DNA was a better competitor for the binding than unmodified DNA. Heterologous DNA that contained no consensus E2F binding motifs also competed well for E2F binding when modified with BPDE. In contrast, transcription factor that does not bend DNA appreciably (GAL4) did not show enhanced affinity for BPDE-modified DNA. These findings suggest that numerous transcription factors that bend DNA may bind with anomalously high affinity to sequences that contain carcinogen-DNA adducts.