Interactions of human replication protein A with oligonucleotides
- Creators
- Kim, Changsoo
- Paulus, Brian F.
- Wold, Marc S.
Abstract
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 RP•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 ~7 x 10^7 M^(-1) for oligo(dT)lO to ~1.5 x 10^(10) M^(-1) for oligo(dT)50. Human RPA binds to oligonucleotides with low cooperativity; the cooperativity parameter (ω) for RPA binding was estimated to be approximately 15.
Additional Information
© 1994 American Chemical Society. Published in print 1 November 1994. This work was supported by Public Health Service Grant GM44721 from the National Institute of Health and General Medicine Institute. We thank L. A. Henricksen, X. Gomes, and Z. Sibenaller for scientific discussions and critical reading of the manuscript. We thank Michael Brenowitz for his help with the initial experiments analyzing RPA binding to oligonucleotides. We also thank Madeline Shea for her assistance and constructive criticism of our data analysis. We thank the University of Iowa DNA Core Facility for oligonucleotide synthesis and the University of Iowa Protein Structure Facility for amino acid analysis.Additional details
- Eprint ID
- 84789
- DOI
- 10.1021/bi00251a031
- Resolver ID
- CaltechAUTHORS:20180212-104556189
- GM44721
- NIH
- Created
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2018-02-13Created from EPrint's datestamp field
- Updated
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2021-11-15Created from EPrint's last_modified field