Recombinant human replication protein A binds to polynucleotides with low cooperativity.
Replication protein A (RPA) is a multisubunit single-stranded DNA-binding protein that is involved in multiple aspects of cellular DNA metabolism. We have determined quantitative estimates of the binding parameters of human replication protein A (hRPA) from equilibrium binding isotherms. The intrinsic binding constant (K) and cooperativity parameter (omega) were determined from analysis of changes in the intrinsic fluorescence of hRPA that occurred upon binding single-stranded DNA homopolynucleotides. The cooperativity of hRPA binding to both poly(dT) and poly(dA) was found to be low (omega = 10-20) at all NaCl concentrations examined (0.3-2 M). In contrast, the apparent binding affinity (K omega) of RPA decreased significantly with increasing salt concentration, such that log [NaCl]/log K omega was -2.8 for poly(dT) and -4.8 for poly(dA). We conclude that the salt dependent decrease in binding affinity resulted from changes in the intrinsic binding constant (K). These data suggest that the interaction of hRPA with single-stranded DNA involves significant electrostatic interactions, similar to other single-stranded DNA-binding proteins. The apparent binding affinity (K omega) of RPA was higher for poly(dT) than for poly(dA); extrapolation of 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).
Kim
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C
Department of Biochemistry, University of Iowa College of Medicine, Iowa City 52242-1109.
Wold
M S
MS
D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004563:Electrochemistry; D006801:Humans; D046911:Macromolecular Substances; D011061:Poly A / Q000378:metabolism; D011071:Poly T / Q000378:metabolism; D011119:Polynucleotides / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011994:Recombinant Proteins / Q000378:metabolism; D051716:Replication Protein A; D012965:Sodium Chloride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D011119:Polynucleotides; C497167:RPA1 protein, human; D011994:Recombinant Proteins; D051716:Replication Protein A; D011061:Poly A; D011071:Poly T; C015465:poly(dA); D012965:Sodium Chloride