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.
Highlights
An early study showing that activityof RNase HI increasesDNA, or if the substrate ahansick at the RNA-DNAjunc-in parallel witDh NA synthesis inbovine lymphocytessuggests tion
From the $Department of Biochemistry and Wancer Center, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642 and §Department of Biochemistry, Wright State University, Dayton, Ohio 45435
RNaseHI from K562 humanerythroleukemia cells. They stranded syntheticDNA template, and subsequentlyex- showed that the enzyme would hydrolyze the 5’phosphodiester tendingtheprimer by DNA polymerization.Thecalf of a single ribonucleotide in a DNA-RNA-DNMDNAheteroduthymusRNase HI makes a structure-specific endonucleolytic cleavage in thReNA primer, releasing it intact, and leaving a mono-ribonucleotide at 5th‘ eterminus of the RNA-DNA junctionT. hisspecificcleavageo, ne nucleotide upstream of the RNA-DNAjunction, is RNA primersequence-andlength-independentC. leavage specificity is lost if theRNA primer is not extended with plex
Summary
DNA, or if the substrate ahansick at the RNA-DNAjunc-in parallel witDh NA synthesis inbovine lymphocytessuggests tion. Since Stein and Hausen (1969) first identified the enzyme, priortojoining of Okazakifragments.The involvement of several different forms of RNaseH have been purified and RNase HIin Okazaki fragment maturation iSnV401 DNArepcharacterized from a number of organisms, including bacteri- lication i n uitro has been suggested (Ishimi et al, 1988; ophage T4 (Hollingsworth and Nossal, 19911, yeast (Kanvanet Waga et al, 1994; Waga and Stillman 1994). In both al., 19831,Drosophila (DiFrancesco and Lehman,19851, Krebs systems, the exact function of RNase HI remains to be elucicells (Cathala et al, 1979),chick embryo
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