Abstract
Eukaryotic replication protein A (RPA) is a single-stranded DNA-binding protein with multiple functions in DNA replication, repair, and genetic recombination. RPA contains an evolutionarily conserved 4-cysteine-type zinc finger motif (X(3)CX(2-4)CX(12-15)CX(2)C) that has a potential role in regulation of DNA replication and repair (Dong, J., Park, J-S., and Lee, S-H. (1999) Biochem. J. 337, 311-317 and Lin, Y.-L., Shivji, M. K. K., Chen, C., Kolodner, R., Wood, R. D., and Dutta, A. (1998) J. Biol. Chem. 273, 1453-1461), even though the zinc finger itself is not essential for its DNA binding activity (Kim, D. K., Stigger, E., and Lee, S.-H. (1996) J. Biol. Chem. 271, 15124-15129). Here, we show that RPA single-stranded DNA (ssDNA) binding activity is regulated by reduction-oxidation (redox) through its zinc finger domain. RPA-ssDNA interaction was stimulated 10-fold by the reducing agent, dithiothreitol (DTT), whereas treatment of RPA with oxidizing agent, diazene dicarboxylic acid bis[N,N-dimethylamide] (diamide), significantly reduced this interaction. The effect of diamide was reversed by the addition of excess DTT, suggesting that RPA ssDNA binding activity is regulated by redox. Redox regulation of RPA-ssDNA interaction was more effective in the presence of 0.2 M NaCl or higher. Cellular redox factor, thioredoxin, was able to replace DTT in stimulation of RPA DNA binding activity, suggesting that redox protein may be involved in RPA modulation in vivo. In contrast to wild-type RPA, zinc finger mutant (cysteine to alanine mutation at amino acid 486) did not require DTT for its ssDNA binding activity and is not affected by redox. Together, these results suggest a novel function for a putative zinc finger in the regulation of RPA DNA binding activity through cellular redox.
Highlights
The replication protein A (RPA1; known as human single-stranded DNA-binding protein) is a three-subunit complex (70, 34, and 11-kDa; p70, p34, and p11, respectively) essential for DNA replication, nucleotide excision repair, and genetic recombination [54]
RPA single-stranded DNA (ssDNA) Binding Activity Is Regulated by Redox—In an effort to understand the regulatory function of RPA, we examined whether RPA ssDNA binding activity is affected by redox
These results strongly suggest that RPA ssDNA binding activity is regulated by redox potential
Summary
The replication protein A (RPA1; known as human single-stranded DNA-binding protein) is a three-subunit complex (70-, 34-, and 11-kDa; p70, p34, and p11, respectively) essential for DNA replication, nucleotide excision repair, and genetic recombination [54]. RPA ssDNA binding activity was sensitive to diamide (Fig. 1b), a chemical that catalyzes the oxidation of free sulfhydryl groups [45], suggesting a possible involvement of cysteine residues in the redox-dependent DNA binding activity of RPA. The inhibitory effect of NEM on RPA ssDNA binding activity strongly indicates that cysteine residues are involved in the redox regulation of RPA DNA binding activity.
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