Cooperative regulation for Okazaki fragment processing by RNase HII and FEN-1 purified from a hyperthermophilic archaeon, Pyrococcus furiosus.
A reconstitution system that recapitulates the processing of Okazaki-primer RNA was established by the heat-stable recombinant enzymes RNase HII and FEN-1 (termed Pf-RNase HII and Pf-FEN-1, respectively) prepared from a hyperthermophilic archaeon, Pyrococcus furiosus. 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 50 degrees C. 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 0.25-5mM MnCl(2), 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.
Sato
Asako
A
Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan.
Kanai
Akio
A
Itaya
Mitsuhiro
M
Tomita
Masaru
M
D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D020027:Pyrococcus furiosus / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016914:Ribonuclease H / Q000737:chemistry*; D013696:Temperature; D013997:Time Factors
D017931:DNA Primers; C022262:Okazaki fragments; D011994:Recombinant Proteins; D012313:RNA; D004247:DNA; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; C091139:ribonuclease HII; D016914:Ribonuclease H