Abstract
Publisher Summary This chapter describes DNA polymerase and discusses the purification of the miniscule activity responsible for DNA synthesis. In the initial search for DNA synthesis in cell-free extracts, DNA was included in the reaction mixture so that it could serve as templates for replication. Several fractions prepared from the crude extract were needed for incorporation of thymidine. Of these, two supplied the nucleases and kinases that generated the missing dNTPs and another the polymerase. From cell extracts prepared by sonic disruption, nucleic acids had to be removed and this was accomplished by a refined precipitation with streptomycin sulfate followed by digestion with DNase. One of the inferences drawn from the replication of a single-stranded, circular template was that DNA polymerase could start a new chain. All three polymerases, although differing significantly in structure, proved to be virtually identical in their mechanisms of DNA synthesis, proofreading, and use of the same building blocks.
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