Recombinant human replication protein A binds to polynucleotides with low cooperativity

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Replication protein A (RPA) is a crucial multisubunit protein essential for various DNA metabolism processes.
• Understanding RPA's DNA-binding characteristics is vital for elucidating its cellular functions.

Purpose of the Study:

• To quantitatively determine the binding parameters of human RPA (hRPA) to single-stranded DNA.
• To investigate the influence of salt concentration on hRPA's DNA binding affinity and cooperativity.

Main Methods:

• Utilized equilibrium binding isotherms to analyze hRPA binding to single-stranded DNA homopolynucleotides (poly(dT) and poly(dA)).
• Measured changes in hRPA's intrinsic fluorescence upon DNA binding to determine binding constants and cooperativity.
• Examined binding parameters across a range of NaCl concentrations (0.3-2 M).

Main Results:

• hRPA exhibited low cooperativity (omega = 10-20) when binding to both poly(dT) and poly(dA) across tested salt concentrations.
• Apparent binding affinity (K omega) significantly decreased with increasing NaCl concentration, indicating salt-dependent interactions.
• The decrease in affinity was primarily attributed to changes in the intrinsic binding constant (K), suggesting significant electrostatic interactions.

Conclusions:

• The interaction between hRPA and single-stranded DNA is characterized by significant electrostatic forces.
• hRPA demonstrates higher binding affinity for poly(dT) than poly(dA), with extrapolated affinities of 1.6 x 10^10 M^-1 and 1.1 x 10^9 M^-1 at 0.2 M NaCl, respectively.