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Recombinant Human Replication Protein A Binds to Polynucleotides with Low Cooperativity

Kim, Changsoo and Wold, Marc S. (1995) Recombinant Human Replication Protein A Binds to Polynucleotides with Low Cooperativity. Biochemistry, 34 (6). pp. 2058-2064. ISSN 0006-2960. doi:10.1021/bi00006a028.

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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 (ω) 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 (ω = 10-20) at all NaCl concentrations examined (0.3-2 M). In contrast, the apparent binding affinity (Kω) of RPA decreased significantly with increasing salt concentration, such that log [NaCl]/log Kω 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 (KO) 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 1O^(1O) M-^1 for poly(dT) and 1.1 x 10^9 M-^1 for poly(dA).

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Additional Information:© 1995 American Chemical Society. Published in print 14 February 1995. This work was supported by U.S. Public Health Service Grant GM44721 from the National Institutes of Health General Medicine Institute. We thank Drs. Paul Mitsis and I. Robert Lehman for communication of results prior to publication and Dr. Paul Mitsis for helpful discussions of fluorescence experiments. We thank the University of Iowa DNA Core Facility for oligonucleotide synthesis.
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Issue or Number:6
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Official Citation:Recombinant Human Replication Protein A Binds to Polynucleotides with Low Cooperativity Changsoo Kim and Marc S. Wold Biochemistry 1995 34 (6), 2058-2064 DOI: 10.1021/bi00006a028
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:84660
Deposited By: Ruth Sustaita
Deposited On:05 Feb 2018 23:52
Last Modified:15 Nov 2021 20:21

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