Zinc affects the conformation of nucleoprotein filaments formed by replication protein A (RPA) and long natural DNA molecules.
Replication protein A is the major single strand DNA binding protein of human cells, composed of three subunits with molecular weights of 70, 32, and 14 kDa. Most of the DNA binding activity of RPA has been mapped to the largest subunit that contains two OB-fold DNA binding domains and a third, OB-like structure in the carboxyterminal domain (CTD). This third domain resembles an OB-fold with a zinc binding domain inserted in the middle of the structure, and has recently been shown to carry a coordinated Zn(II) ion. The bound metal ion is essential for the tertiary structure of the RPA70-CTD, and appears to modulate its DNA binding activity when tested with synthetic oligonucleotides. We show here that zinc strongly affects the conformation of nucleoprotein filaments formed between RPA and long natural DNA molecules. In these experiments, the CTD is dispensable for DNA binding and the unwinding of long double stranded DNA molecules. However, using band shift assays and electron microscopy, we found that RPA-DNA complexes contract at zinc concentrations that do not affect the conformations of complexes formed between DNA and a RPA70 deletion construct lacking the CTD. Our data suggest that nucleoprotein complexes with RPA in its natural, zinc-bearing form may have a compact rather than an extended conformation.
Eckerich
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Department of Biology, University of Konstanz, Germany. carmen.eckerich@uni-konstanz.de
Fackelmayer
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Knippers
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D017104:Bacteriophage M13; D002413:Cations, Divalent; D004247:DNA / Q000737:chemistry*; D004265:DNA Helicases / Q000737:chemistry; D018076:DNA, Complementary / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004587:Electrophoresis, Agar Gel; D006801:Humans; D007382:Intermediate Filaments / Q000737:chemistry* / Q000648:ultrastructure; D008854:Microscopy, Electron; D009698:Nucleoproteins / Q000737:chemistry* / Q000648:ultrastructure; D011487:Protein Conformation / Q000187:drug effects*; D051716:Replication Protein A; D015032:Zinc / Q000494:pharmacology*
D002413:Cations, Divalent; D018076:DNA, Complementary; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009698:Nucleoproteins; C497167:RPA1 protein, human; D051716:Replication Protein A; D004247:DNA; D004265:DNA Helicases; D015032:Zinc