Quantification of DNA-protein interaction by UV crosslinking

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Protein-DNA interactions are crucial for gene transcription regulation.
• Accurate measurement of protein binding affinity to specific DNA sequences is essential.
• Existing methods may have limitations in quantifying DNA binding proteins.

Purpose of the Study:

• To introduce and validate the DNA protein crosslinking (DPC) assay for measuring DNA binding protein characteristics.
• To determine the affinity, amount, and molecular weight of DNA binding proteins.
• To assess the utility of the DPC assay in evaluating proteins' potential to regulate transcription.

Main Methods:

• Application of the DNA protein crosslinking (DPC) assay to nuclear extracts and purified proteins.
• Theoretical analysis of binding data to calculate affinity constants.
• Measurement of protein binding to specific (promoter) and non-specific DNA sequences.

Main Results:

• The DPC assay successfully measured affinity, amount, and molecular weight of DNA binding proteins.
• Two proteins (DPC80 and DPC107) exhibited high affinity (2 x 10(-9) M) for the mouse thymidine kinase promoter MT3 region, 10^4 times higher than for non-specific DNA.
• Crosslinking efficiency was determined, enabling estimation of binding protein abundance.

Conclusions:

• The DPC assay is a reliable method for quantifying DNA binding proteins.
• The assay provides valuable insights into protein-DNA interactions and their role in transcription regulation.
• DPC assay facilitates accurate assessment of DNA binding proteins' influence on gene expression.