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Polynucleotide polymerases, or nucleotidyl transferases, are enzymes that catalyze the template-instructed polymerization of deoxyribo- or ribonu-cleoside triphosphates into polymeric nucleic acid - DNA or RNA. Depending on their substrate specificity, polymerases are classed as RNA- or DNA-dependent polymerases which copy their templates into RNA or DNA (all combinations of substrates are possible). Polymerization, or nucleotidyl transfer, involves formation of a phosphodiester bond that results from nu-cleophilic attack of the 3 '-OH of primer-template on the a-phosphate group of the incoming nucleoside triphosphate. Although substantial diversity of sequence and function is observed for natural polymerases, there is evidence that many employ the same mechanism for DNA or RNA synthesis. On the basis of the crystal structures of polymerase replication complexes, a ''two-metal-ion mechanism'' of nucleotide addition was proposed [1]; during this two divalent metal ions stabilize the structure and charge of the expected pentacovalent transition state (Figure B.16.1).

Since the discovery of Escherichia coli DNA polymerase I in 1957 [2], many polymerases have been identified in prokaryotes and eukaryotes, including the recent discovery of several error-prone DNA polymerases [3]. Primary sequence alignments revealed that these polymerases can be cate-

4.1 Selectivity of DNA Replication 3'-

5' template base base

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