Single-strand DNA binding protein from rat liver: interactions with supercoiled DNA

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Single-strand DNA binding proteins play crucial roles in DNA replication, repair, and recombination.
• Understanding protein-DNA interactions is fundamental to deciphering cellular processes.

Purpose of the Study:

• To investigate the binding characteristics of the single-strand DNA binding protein S25 from normal rat liver.
• To elucidate the structural organization and DNA interaction mechanisms of S25.

Main Methods:

• Competition experiments to assess DNA binding preference.
• Sedimentation analysis and nitrocellulose filter assays for binding kinetics.
• Cross-linking with glutaraldehyde and dimethyl suberimidate to study complex structure.
• Micrococcal nuclease assays to evaluate DNA accessibility.
• Enzymatic assays with nicking-closing enzyme to assess DNA structural changes.

Main Results:

• S25 exhibits preferential binding to supercoiled DNA over relaxed DNA duplexes.
• Non-cooperative binding of S25 to SV40 supercoiled DNA (FI) was observed, reaching saturation at a protein to DNA weight ratio of approximately 2.
• Glutaraldehyde fixation revealed beaded structures of S25 on SV40 DNA, with 14-16 beads per molecule.
• Dimethyl suberimidate cross-linking indicated the presence of S25 oligomers up to twenty monomers.
• Complexes treated with glutaraldehyde showed 10% of the genome resistant to micrococcal nuclease.
• S25 association with SV40 DNA induced superhelical forms in the presence of a nicking-closing enzyme, altering DNA helical structure.

Conclusions:

• S25 is a DNA-binding protein with a preference for supercoiled DNA structures.
• The protein forms distinct oligomeric structures on DNA, influencing DNA accessibility and helical conformation.
• These findings provide insights into the structural role of S25 in modulating DNA topology.