Interaction of the p70 subunit of RPA with a DNA template directs p32 to the 3 '-end of nascent DNA

Human replication protein A is a heterotrimeric protein involved in various processes of DNA metabolism. To understand the contribution of replication protein A individual subunits to DNA binding, we have expressed them separ ately as soluble maltose binding protein fusion proteins. Using a DNA const ruct that had a photoreactive group incorporated at the 3'-end of the prime r strand, we show that the p70 subunit on its own is efficiently cross-link ed to the primer at physiological concentrations. In contrast, crosslinking of the p32 subunit required two orders of magnitude higher protein concent rations. In no case was the p14 subunit labelled above background. p70 seem s to be the predominant subunit to bind single-stranded DNA and this intera ction positions the p32 subunit to the 3'-end of the primer. (C) 1999 Feder ation of European Biochemical Societies.