Anti-RFA2 (aa 115-129) polyclonal antibody

An encounter between a DNA-translocating enzyme and a DNA-bound protein must occur frequently in the cell, but little is known about its outcome. Here we developed a multicolor single-molecule fluorescence approach to simultaneously monitor single-stranded DNA (ssDNA) translocation by a helicase, and the fate of another protein bound to the same DNA. Distance-dependent fluorescence quenching by the iron-sulfur cluster of the archaeal XPD (Rad3) helicase was used as a calibrated proximity signal. Despite the similar equilibrium DNA-binding properties, the two cognate ssDNA-binding proteins RPA1 and RPA2 differentially affected XPD translocation. RPA1 competed with XPD for ssDNA access. In contrast, RPA2 did not interfere with XPD-ssDNA binding but markedly slowed down XPD translocation. Mechanistic models of bypassing DNA-bound proteins by the Rad3 family helicases and their biological implications are discussed.

Replication protein A (RPA) is a mammalian single-stranded DNA binding factor essential for DNA replication, repair, and recombination. It is composed of three subunits of 70, 34, and 13 kDa (Rpa1, Rpa2, and Rpa3, respectively), Deletion mapping of the Rpa2 subunit identified the domain required for interaction with Rpa1 and Rpa3 which does not include the N-terminal domain that is phosphorylated during S phase, Deletion mapping of Rpa1 defined three domains. The C-terminal third of the Rpa1 polypeptide binds Rpa2 which itself forms a bridge between Rpa1 and Rpa3, The N-terminal third of Rpa1 bound single-stranded DNA under low stringency conditions only (0.1 M NaCl), while a central domain binds to single-stranded DNA under both low and high stringency conditions (0.5 M NaCl), Binding to p53 requires the N-terminal third of Rpa1 with some contribution from the C-terminal third. The evolutionarily conserved putative zinc finger near the C terminus of Rpa1 was not required for binding to single-stranded DNA, Rpa2, or p53, However, all three subdomains of Rpa1 and the zinc finger were essential for supporting DNA replication in vitro. These experiments are a first step toward defining peptide components responsible for the many functions of the RPA protein complex.