Zinc finger of replication protein A, a non-DNA binding element, regulates its DNA binding activity through redox.
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.
Park
J S
JS
Department of Biochemistry, the Indiana University Cancer Center, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Wang
M
M
Park
S J
SJ
Lee
S H
SH
D000595:Amino Acid Sequence; D002614:Chelating Agents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D004265:DNA Helicases; D004277:DNA, Single-Stranded / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism; D003958:Diamide; D004229:Dithiothreitol; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D010618:Phenanthrolines / Q000494:pharmacology; D011506:Proteins / Q000737:chemistry / Q000235:genetics*; D013439:Sulfhydryl Reagents; D013879:Thioredoxins / Q000494:pharmacology; D015534:Trans-Activators; D016335:Zinc Fingers
D002614:Chelating Agents; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D010618:Phenanthrolines; D011506:Proteins; D013439:Sulfhydryl Reagents; D015534:Trans-Activators; C045264:replication initiator protein; D003958:Diamide; D013879:Thioredoxins; D004265:DNA Helicases; D003545:Cysteine; D004229:Dithiothreitol; C025205:1,10-phenanthroline