Structure-specific cleavage of the RNA primer from Okazaki fragments by calf thymus RNase HI

Lin Huang, Yong Kim, John Turchi, Robert A. Bambara

Research output: Contribution to journalArticle

29 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)25922-25927
Number of pages6
JournalJournal of Biological Chemistry
Volume269
Issue number41
StatePublished - Oct 14 1994
Externally publishedYes

Fingerprint

Thymus
Thymus Gland
RNA
DNA
Ribonucleotides
RNA Cleavage
Ribonuclease H
HIV Reverse Transcriptase
DNA Primers
Single-Stranded DNA
DNA Replication
Polymerization
Nucleotides
Substrates
Transcription
Okazaki fragments
RNA primers
ribonuclease HI
Escherichia coli
Annealing

ASJC Scopus subject areas

  • Biochemistry

Cite this

Structure-specific cleavage of the RNA primer from Okazaki fragments by calf thymus RNase HI. / Huang, Lin; Kim, Yong; Turchi, John; Bambara, Robert A.

In: Journal of Biological Chemistry, Vol. 269, No. 41, 14.10.1994, p. 25922-25927.

Research output: Contribution to journalArticle

Huang, Lin ; Kim, Yong ; Turchi, John ; Bambara, Robert A. / Structure-specific cleavage of the RNA primer from Okazaki fragments by calf thymus RNase HI. In: Journal of Biological Chemistry. 1994 ; Vol. 269, No. 41. pp. 25922-25927.
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