Interactions of human replication protein A with oligonucleotides.
Replication protein A (RPA) is a heterotrimeric, single-stranded DNA binding protein that is essential for eukaryotic DNA replication. In order to gain a better understanding of the interactions between RPA and DNA, we have examined the interactions of human RPA with single-stranded oligonucleotides. Our analysis of RPA.DNA complexes demonstrated that RPA binds as a heterotrimer. Stoichiometric binding reactions monitored by fluorescence quenching indicated that the binding site size of human RPA is 30 nucleotides and that between 20-30 nucleotides of DNA directly interact with RPA. The binding of RPA to DNA of different lengths was systematically examined using deoxythymidine-containing oligonucleotides. We found that the binding affinity of RPA for short oligonucleotides was length dependent. The apparent association constant of RPA varied over 200-fold from approximately 7 x 10(7) M-1 for oligo(dT)10 to approximately 1.5 x 10(10) M-1 for oligo(dT)50. Human RPA binds to oligonucleotides with low cooperativity; the cooperativity parameter (omega) for RPA binding was estimated to be approximately 15.
Kim
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Department of Biochemistry, University of Iowa College of Medicine, Iowa City 52242.
Paulus
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Wold
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MS
D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D002499:Centrifugation, Density Gradient; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells / Q000737:chemistry; D006358:Hot Temperature; D006801:Humans; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000378:metabolism; D051716:Replication Protein A; D012965:Sodium Chloride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; C497167:RPA1 protein, human; D051716:Replication Protein A; C027903:oligo (dT); D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D004247:DNA; D011116:Polynucleotide 5'-Hydroxyl-Kinase