RECOMBINANT HUMAN REPLICATION PROTEIN-A BINDS TO POLYNUCLEOTIDES WITHLOW COOPERATIVITY

Replication protein A (RPA) is a multisubunit single-stranded DNA-bind ing protein that is involved in multiple aspects of cellular DNA metab olism. We have determined quantitative estimates of the binding parame ters of human replication protein A (hRPA) from equilibrium binding is otherms. The intrinsic binding constant (K) and cooperativity paramete r (omega) were determined from analysis of changes in the intrinsic fl uorescence of hRPA that occurred upon binding single-stranded DNA homo polynucleotides. The cooperativity of hRPA binding to both poly(dT) an d poly(dA) was found to be low (omega = 10-20) at all NaCl concentrati ons examined (0.3-2 M). In contrast, the apparent binding affinity (K omega) of RPA decreased significantly with increasing salt concentrati on, such that log [NaCl]/log K omega was -2.8 for poly(dT) and -4.8 fo r poly(dA). We conclude that the salt dependent decrease in binding af finity resulted from changes in the intrinsic binding constant (K). Th ese data suggest that the interaction of hRPA with single-stranded DNA involves significant electrostatic interactions, similar to other sin gle-stranded DNA-binding proteins. The apparent binding affinity (K om ega) of RPA was higher for poly(dT) than for poly(dA); extrapolation o f our data indicated that the apparent binding affinity at 0.2 M NaCl was 1.6 x 10(10) M(-1) for poly(dT) and 1.1 x 10(9) M(-1) for poly(dA) .