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High-affinity binding of the cell cycle-regulated transcription factors E2F1 and E2F4 to benzo[a]pyrene diol
D G Johnson1, A Coleman, K L Powell
1University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville 78957, USA.
Abstract:
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 than for 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.
Insights
Carcinogen-DNA adducts, like those from benzo[a]pyrene diol epoxide (BPDE), increase the binding affinity of DNA-bending transcription factors, such as E2F. This suggests a mechanism for how DNA damage impacts gene regulation.
Area of Science:
- Molecular Biology
- Carcinogenesis
- Gene Regulation
Background:
- Benzo[a]pyrene diol epoxide (BPDE) forms DNA adducts that can alter transcription factor binding.
- Previous studies suggested BPDE-induced DNA bends increase Sp1 transcription factor affinity.
- The transcription factor E2F is known to bend DNA upon binding.
Purpose of the Study:
- To investigate if BPDE modification of DNA affects the binding affinity of the transcription factor E2F.
- To determine if DNA bending is a key factor in enhanced transcription factor binding to damaged DNA.
Main Methods:
- Gel mobility-shift assays were used to assess E2F binding to DNA.
- Partially purified and recombinant human E2F1, E2F4, and DP1 proteins were utilized.
- DNA fragments, both modified and unmodified by BPDE, were tested for binding competition.
Main Results:
- E2F proteins (E2F1/DP1 and E2F4/DP1) showed significantly higher apparent affinity for BPDE-modified DNA compared to unmodified DNA.
- BPDE-modified DNA, even without consensus E2F sites, effectively competed for E2F binding.
- A non-DNA-bending transcription factor (GAL4) did not exhibit enhanced affinity for BPDE-modified DNA.
Conclusions:
- DNA damage by BPDE enhances the binding affinity of DNA-bending transcription factors like E2F.
- The ability of a transcription factor to bend DNA appears crucial for this anomalous high-affinity binding to adducts.
- Carcinogen-DNA adducts may broadly influence gene regulation by altering the binding of various DNA-bending transcription factors.