HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 276, Issue 39, 36446-36453, September 28, 2001

This Article Full Text Full Text (PDF) All Versions of this Article: 276/39/36446    most recent M104386200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bastin-Shanower, S. A. Articles by Brill, S. J. Search for Related Content PubMed PubMed Citation Articles by Bastin-Shanower, S. A. Articles by Brill, S. J.

Functional Analysis of the Four DNA Binding Domains of Replication Protein A
THE ROLE OF RPA2 IN ssDNA BINDING^*

Suzanne A. Bastin-Shanower and Steven J. Brill

From the Department of Molecular Biology and Biochemistry, Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey 08854

Replication Protein A (RPA), the heterotrimeric single-stranded DNA (ssDNA)-binding protein of eukaryotes, contains four ssDNA binding domains (DBDs) within its two largest subunits, RPA1 and RPA2. We analyzed the contribution of the four DBDs to ssDNA binding affinity by assaying recombinant yeast RPA in which a single DBD (A, B, C, or D) was inactive. Inactivation was accomplished by mutating the two conserved aromatic stacking residues present in each DBD. Mutation of domain A had the most severe effect and eliminated binding to a short substrate such as (dT)12. RPA containing mutations in DBDs B and C bound to substrates (dT)12, 17, and 23 but with reduced affinity compared with wild type RPA. Mutation of DBD-D had little or no effect on the binding of RPA to these substrates. However, mutations in domain D did affect the binding to oligonucleotides larger than 23 nucleotides (nt). Protein-DNA cross-linking indicated that DBD-A (in RPA1) is essential for RPA1 to interact efficiently with substrates of 12 nt or less and that DBD-D (RPA2) interacts efficiently with oligonucleotides of 27 nt or larger. The data support a sequential model of binding in which DBD-A is responsible for the initial interaction with ssDNA, that domains A, B, and C (RPA1) contact 12-23 nt of ssDNA, and that DBD-D (RPA2) is needed for RPA to interact with substrates that are 23-27 nt in length.

This article has been cited by other articles:

				E. Fanning, V. Klimovich, and A. R. Nager A dynamic model for replication protein A (RPA) function in DNA processing pathways Nucleic Acids Res., September 10, 2006; 34(15): 4126 - 4137. [Abstract] [Full Text] [PDF]

				A. J. Bartrand, D. Iyasu, S. M. Marinco, and G. S. Brush Evidence of Meiotic Crossover Control in Saccharomyces cerevisiae Through Mec1-Mediated Phosphorylation of Replication Protein A Genetics, January 1, 2006; 172(1): 27 - 39. [Abstract] [Full Text] [PDF]

				C.-J. Park, J.-H. Lee, and B.-S. Choi Solution structure of the DNA-binding domain of RPA from Saccharomyces cerevisiae and its interaction with single-stranded DNA and SV40 T antigen Nucleic Acids Res., July 25, 2005; 33(13): 4172 - 4181. [Abstract] [Full Text] [PDF]

				A. A. Franco, W. M. Lam, P. M. Burgers, and P. D. Kaufman Histone deposition protein Asf1 maintains DNA replisome integrity and interacts with replication factor C Genes & Dev., June 1, 2005; 19(11): 1365 - 1375. [Abstract] [Full Text] [PDF]

				A. J. Bartrand, D. Iyasu, and G. S. Brush DNA Stimulates Mec1-mediated Phosphorylation of Replication Protein A J. Biol. Chem., June 18, 2004; 279(25): 26762 - 26767. [Abstract] [Full Text] [PDF]

				J. E. Nuss and G. M. Alter Denaturation of replication protein A reveals an alternative conformation with intact domain structure and oligonucleotide binding activity Protein Sci., May 1, 2004; 13(5): 1365 - 1378. [Abstract] [Full Text] [PDF]

				P. E. Pestryakov, D. Y. Khlimankov, E. Bochkareva, A. Bochkarev, and O. I. Lavrik Human replication protein A (RPA) binds a primer-template junction in the absence of its major ssDNA-binding domains Nucleic Acids Res., March 26, 2004; 32(6): 1894 - 1903. [Abstract] [Full Text] [PDF]

				S. Falt, K. Holmberg, B. Lambert, and A. Wennborg Long-term global gene expression patterns in irradiated human lymphocytes Carcinogenesis, November 1, 2003; 24(11): 1837 - 1845. [Abstract] [Full Text] [PDF]

				A. I. Arunkumar, M. E. Stauffer, E. Bochkareva, A. Bochkarev, and W. J. Chazin Independent and Coordinated Functions of Replication Protein A Tandem High Affinity Single-stranded DNA Binding Domains J. Biol. Chem., October 17, 2003; 278(42): 41077 - 41082. [Abstract] [Full Text] [PDF]

				P. E. Pestryakov, K. Weisshart, B. Schlott, S. N. Khodyreva, E. Kremmer, F. Grosse, O. I. Lavrik, and H.-P. Nasheuer Human Replication Protein A. THE C-TERMINAL RPA70 AND THE CENTRAL RPA32 DOMAINS ARE INVOLVED IN THE INTERACTIONS WITH THE 3'-END OF A PRIMER-TEMPLATE DNA J. Biol. Chem., May 2, 2003; 278(19): 17515 - 17524. [Abstract] [Full Text] [PDF]

				S. Bhattacharya, M.-V. Botuyan, F. Hsu, X. Shan, A.I. Arunkumar, C. H. Arrowsmith, A. M. Edwards, and W. J. Chazin Characterization of binding-induced changes in dynamics suggests a model for sequence-nonspecific binding of ssDNA by replication protein A Protein Sci., October 1, 2002; 11(10): 2316 - 2325. [Abstract] [Full Text] [PDF]

				H. Yang, P. D. Jeffrey, J. Miller, E. Kinnucan, Y. Sun, N. H. Thoma, N. Zheng, P.-L. Chen, W.-H. Lee, and N. P. Pavletich BRCA2 Function in DNA Binding and Recombination from a BRCA2-DSS1-ssDNA Structure Science, September 13, 2002; 297(5588): 1837 - 1848. [Abstract] [Full Text] [PDF]

				F. Marini and R. D. Wood A Human DNA Helicase Homologous to the DNA Cross-link Sensitivity Protein Mus308 J. Biol. Chem., March 1, 2002; 277(10): 8716 - 8723. [Abstract] [Full Text] [PDF]