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Source: After Cbargaff E. et ai.1S49. J Biul Chem 177. 405.

Source: After Cbargaff E. et ai.1S49. J Biul Chem 177. 405.

Finding the Polymerases that Make DNA

Rigorous proof that a single DNA chain is the template that directs the synthesis of a complementary DNA chain had to await the development of test-tube (in vitro) systems for DNA synthesis. These came much Faster than anticipated by molecular geneticists, whose world until then had been far removed from that of the biochemist well versed in the procedures needed for enzyme isolation. Leading this biochemical assault on DNA replication was U.S. biochemist Arthur Romberg, who by 1956 had demonstrated DNA synthesis in cell-free extracts of bacteria. Over the next several years, Kornberg went on to show that a specific polymerizing enzyme was needed to catalyze the linking together of the building-block precursors of DNA. Kornberg's studies revealed that the nucleotide building blocks for DNA are energy-rich precursors (dATP, dGTP, dCTP, and dTTP; Figure 2-7). Further studies identified a single polypeptide, DNA polymerase I (DNA Pol I), that was capable of catalyzing the synthesis of new DNA strands. It links the nucleotide precursors by 3' —* 5' phos-phodiester bonds (Figure 2-8). Furthermore, it works only in the presence of DNA, which is needed to order the four nunleolides in the polynucleotide product.

DNA Pol J depends on a DNA template to determine the sequence of the DNA it is synthesizing. This was first demonstrated by allowing the enzyme to work in the presence of DNA molecules that contained varying amounts of A:T and G:C base pairs. In every case, the enzymatically synthesized product had the base ratios of the template DNA (Table 2-1). During this cell-free synthesis, no synthesis of proteins or any other molecular class occurs, unambiguously eliminating any non-DNA compounds as intermediate carriers of genetic nucleoside:

pyriniltltm* trîrse deoxycyttdine r—"— l cytosine (DNA) \>

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