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The Initial Step of in Vitro Synthesis of Deoxyribonucleic Acid by the T4 Deoxyribonucleic Acid Polymerase

From the ¹ From the Department of Physiological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

In the initial step of the Goulian-Lucas-Kornberg model for the in vitro synthesis of DNA by the T4 DNA polymerase, a short sequence of nucleotides at the 3' terminus of the single stranded DNA substrate base pairs with an internal nucleotide sequence of the same strand, forming a loop. With the use of the 3' terminus as a primer, the polymerase then replicates the rest of the strand to form a hairpin structure. In this communication, evidence for a refinement of this model is presented. It appears that the loop is formed by base pairing of two internal sequences, one near the 3' terminus and one further back on the same strand. The polymerase then binds to the unpaired 3' terminus and by virtue of its hydrolytic activity degrades it exonucleolytically. When the polymerase reaches the base-paired region it reverses direction and begins incorporating nucleotides, finally yielding the hairpin structure.

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