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 hum an 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 larg est subunit that contains two OB-fold DNA binding domains and a third, OB-l ike structure in the carboxyterminal domain (CTD). This third domain resemb les an OB-fold with a zinc binding domain inserted in the middle of the str ucture, and has recently been shown to early a coordinated Zn(II) ion. The bound metal ion is essential for the tertiary structure of the RPA70-CTD, a nd 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 molecule s. In these experiments, the CTD is dispensable for DNA binding and the unw inding of long double stranded DNA molecules. However, using band shift ass ays and electron microscopy, we found that RPA-DNA complexes contract at zi nc concentrations that do not affect the conformations of complexes formed between DNA and a RPA70 deletion construct lacking the CTD. Our data sugges t that nucleoprotein complexes with RPA in its natural, zinc-bearing form m ay have a compact rather than an extended conformation. (C) 2001 Elsevier S cience B.V. All rights reserved.