DNA Synthesis Catalyzed in vitro by Yeast Extracts

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Abstract
A procedure to prepare crude extracts from single colonies of S. cerevisiae is described. Partially purified extracts catalyzed DNA synthesis directed by single-stranded [phage] fd DNA. Maximum activity requires the presence of ribonucleoside triphosphates although extensive DNA synthesis is observed in the presence of only deoxynucleoside triphosphates. Alkaline sucrose gradient analysis demonstrated that initiation of new DNA chains occurs in vitro and the DNA products synthesized are heterogeneous in size. Isopycnic analysis of the products of ribonucleotide-initiated fd DNA replication showed covalent linkage between the initiator RNA and the newly synthesized DNA. The fd-replicating capacity of extracts prepared from cell-division-cycle mutants, defective in events controlling DNA initiation or elongation, showed an increased thermosensitivity in the DNA replication reaction in vitro.