title: Structure-specific cleavage of the RNA primer from Okazaki fragments by calf thymus RNase HI.
abstract: Cleavage specificity of RNase HI was examined on model Okazaki fragments, to determine the likely role of this nuclease in lagging strand DNA replication. Each substrate was prepared by annealing a short RNA primer, made by transcription in vitro, to a single-stranded synthetic DNA template, and subsequently extending the primer by DNA polymerization. The calf thymus RNase HI makes a structure-specific endonucleolytic cleavage in the RNA primer, releasing it intact, and leaving a mono-ribonucleotide at the 5' terminus of the RNA-DNA junction. This specific cleavage, one nucleotide upstream of the RNA-DNA junction, is RNA primer sequence- and length-independent. Cleavage specificity is lost if the RNA primer is not extended with DNA, or if the substrate has a nick at the RNA-DNA junction. In addition, the cleavage at a single site requires Mg2+. Cleavage in the presence of Mn2+ is less specific. Neither human immunodeficiency virus reverse transcriptase nor Escherichia coli RNases H perform such a structure-specific cleavage before an RNA-DNA junction. Our work indicates that calf RNase HI is designed to recognize Okazaki fragments. It has the specificity to remove their initiator RNA segments, except for one ribonucleotide, by a single endonucleolytic cleavage in vivo.
authors.affiliation: Department of Biochemistry, University of Rochester, School of Medicine and Dentistry, New York 14642.
authors.forename: L
authors.initials: L
authors.lastname: Huang
authors.forename: Y
authors.initials: Y
authors.lastname: Kim
authors.forename: J J
authors.initials: JJ
authors.lastname: Turchi
authors.forename: R A
authors.initials: RA
authors.lastname: Bambara
mesh_terms: D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004247:DNA / Q000378:metabolism*; D004261:DNA Replication; D006678:HIV / Q000201:enzymology; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D012313:RNA / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D016914:Ribonuclease H / Q000187:drug effects / Q000378:metabolism*; D013379:Substrate Specificity; D013950:Thymus Gland / Q000201:enzymology*
chemical_list: C022262:Okazaki fragments; C034477:RNA primers; D008345:Manganese; D012313:RNA; D004247:DNA; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H; C078856:ribonuclease HI; D008274:Magnesium