Enzymatic completion of mammalian lagging-strand DNA replication

John J. Turchi, Lin Huang, Richard S. Murante, Yong Kim, Robert A. Bambara

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Abstract

Using purified proteins from calf and a synthetic substrate, we have reconstituted the enzymatic reactions required for mammalian Okazaki fragment processing in vitro. The required reactions are removal of initiator RNA, synthesis from an upstream fragment to generate a nick, and then ligation. With our substrate, RNase H type I (RNase HI) makes a single cut in the initiator RNA, one nucleotide 5' of the RNA-DNA junction. The double strand specific 5' to 3' exonuclease removes the remaining monoribonucleotide. After dissociation of cleaved RNA, synthesis by DNA polymerase generates a nick, which is then sealed by DNA ligase I. The unique specificities of the two nucleases for primers with initiator RNA strongly suggest that they perform the same reactions in vivo.

Original languageEnglish (US)
Pages (from-to)9803-9807
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume91
Issue number21
DOIs
StatePublished - Oct 11 1994

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