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Incorporation of 5-bromodeoxycytidine in the adenovirus 2 replication origin interferes with nuclear factor 1 binding
E de Vries1, S M Bloemers, P C van der Vliet
1Laboratory for Physiological Chemistry, State University of Utrecht, The Netherlands.
Abstract:
We have studied the binding of nuclear factor 1 (NFI), a human sequence-specific DNA-binding protein, to a DNA fragment substituted in vitro with 5-bromodeoxycytidine (5-BrdC). Even at low substitution grades binding of NFI to its recognition sequence was considerably lower than with the unsubstituted control fragment. We developed a procedure to cleave substituted DNA specifically at a BrdC residue and searched for contacts between NFI and 5-BrdC residues by an interference assay. Surprisingly, no specific contacts were found in or near the recognition sequence. It appeared instead that interference was inversely related to the distance of a 5-BrdC residue from the NFI binding site. Models to explain these results, including a possible sliding mechanism, are discussed.
Insights
Nuclear factor 1 (NFI) binding to DNA decreases with 5-bromodeoxycytidine (5-BrdC) substitution. Surprisingly, NFI does not directly contact 5-BrdC within its recognition sequence, suggesting an indirect interaction mechanism.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Nuclear factor 1 (NFI) is a human transcription factor that binds specific DNA sequences.
- 5-bromodeoxycytidine (5-BrdC) is a modified nucleoside that can be incorporated into DNA.
- Understanding protein-DNA interactions is crucial for gene regulation.
Purpose of the Study:
- To investigate the effect of 5-BrdC substitution on NFI DNA binding.
- To identify specific contacts between NFI and 5-BrdC residues.
- To elucidate the mechanism by which 5-BrdC affects NFI binding.
Main Methods:
- In vitro DNA binding assays using NFI and 5-BrdC substituted DNA fragments.
- Development of a specific DNA cleavage procedure at 5-BrdC residues.
- Interference assay to detect protein-DNA contacts.
Main Results:
- NFI binding affinity to its recognition sequence was significantly reduced by 5-BrdC substitution, even at low substitution levels.
- No direct contacts were detected between NFI and 5-BrdC residues within or near the NFI recognition site.
- The degree of interference with NFI binding was inversely proportional to the distance of the 5-BrdC residue from the NFI binding site.
Conclusions:
- 5-BrdC incorporation into DNA inhibits NFI binding, but not through direct contact within the recognition sequence.
- The observed inhibition suggests an indirect mechanism, potentially involving DNA flexibility or a sliding interaction.
- Further research is needed to fully understand the implications of modified DNA bases on transcription factor binding and function.