A reconstitution system that recapitulates the processing of Okazaki-primer RNA was established by the heat-stable recombinant enzymes RNase HII and FEN-I (termed Pf-RNase HII and Pf-FEN-1, respectively) prepared from a hyperthermophilic archaeon, Pyrococcus fitriosus. A 35-mer RNA-DNA/DNA hybrid substrate mimicking an Okazaki fragment was used to investigate the properties of the processing reaction in vitro at 50degreesC. Pf-RNase HII endonucleolytically cleaves the RNA primer region, but does not cut the junction between RNA and DNA. Removal of the RNA of the RNA-DNA junction was brought about by Pf-FEN-1 after Pf-RNase HII digestion. In the presence of 0.25-5 mM MnCl2, Pf-FEN-1 alone weakly cleaved the junction. The addition of Pf-RNase HII to the reaction mixture increased removal efficiency and optimal Pf-FEN-1 activity was achieved at an equal amount of the two enzymes. These results indicate that there are at least two steps in the degradation of primer RNA requiring a step-specific enzyme. It is likely that Pf-RNase HII and Pf-FEN-1 cooperatively process Okazaki fragment during lagging-strand DNA replication. (C) 2003 Elsevier Inc. All rights reserved.