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J Biol Chem 248: 1361-4 (1973)[73097107]

Initiation of deoxyribonucleic acid synthesis. IV. Incorporation of the ribonucleic acid primer into the phage replicative form.

O. Westergaard, D. Brutlag & A. Kornberg

The product of in vitro replication of the ØX174 or M13 viral strand is known to be a circular duplex with a discontinuity in the synthetic strand (RF II). This replication intermediate has now been converted to an alkali-stable covalent duplex (RF I) through the joint action of Escherichia Coli DNA polymerase I and E. coli ligase. Failure of T4 DNA polymerase to substitute for the E. coli enzyme implied the presence of an RNA priming segment at the 5' end which the unique 5' => 3' exonuclease function of the E. coli enzyme can excise. This suggestion was confirmed by conversion of the RF II product to an alkali-labile RF I through the joint action of T4 DNA polymerase and T4 ligase, an enzyme which can join RNA ends, and by location of the alkali-labile link-age of the RF I in the complementary (synthetic) strand. These results extend earlier evidence for RNA priming of DNA replication. They also call attention to a possible physiological role of E. coli DNA polymerase I in the excision of such RNA primers during DNA replication.

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