Anomalous DNA binding by E2 regulatory protein driven by spacer sequence TATA

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Human papillomavirus (HPV) regulatory protein E2 plays a crucial role in viral gene expression.
• Understanding the DNA-binding specificities of E2 is essential for deciphering viral replication and pathogenesis.
• Anomalously weak binding of E2 to certain DNA sequences, like those containing TATA, requires detailed investigation.

Purpose of the Study:

• To investigate the molecular mechanisms behind the weak binding of HPV E2 protein to DNA sequences containing the TATA spacer.
• To elucidate the role of divalent cations, specifically magnesium (Mg2+) and calcium (Ca2+), in modulating the DNA-E2 interaction.
• To determine how different ion concentrations and types affect the binding affinity and DNA conformation.

Main Methods:

• DNase I footprinting assays to assess protein-DNA interactions in the presence of Mg2+ and Ca2+.
• Cation titration experiments to study the dependence of DNA-E2 binding affinities on ion concentration.
• Comparative binding studies of TATA and ATAT DNA sequences in various ionic conditions (K+, Na+, Ca2+, Mg2+).

Main Results:

• DNase I cutting was reduced at CpG sites near the TATA sequence, indicating E2 binding.
• The TATA sequence released approximately twice as many Mg2+ ions upon E2 binding compared to other spacer sequences.
• E2 affinity for TATA relative to ATAT was nearly equal in K+ but decreased with increasing cation charge density (K+ < Na+ < Ca2+ < Mg2+).
• Mg2+ significantly weakened E2 affinity for TATA, especially at higher concentrations, suggesting additional ion binding and DNA distortion.

Conclusions:

• The TATA spacer sequence can adopt a DNA conformation that inherently binds HPV E2 protein weakly.
• Divalent cations, particularly Mg2+, exacerbate this weak binding by distorting the E2 binding site and requiring displacement of additional bound ions.
• These findings provide insights into sequence-specific DNA recognition by viral proteins and the influence of ionic environments.