Polynucleotide polymerases

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- 

A host of detection principles can be used to sense the presence of double-stranded DNA. Fluorescent detection techniques employ dyes that change fluorescence properties upon binding to DNA (this is also discussed in detail in Chapter 2.5). 

---

Figure 13 7 The two-metal-ion mechanism for polynucleotide polymerases. One metal ion (usually Mg2+) activates the 3 -OH group of the primer terminus and stabilizes one of the partly negatively charged equatorial oxygen atoms of the phosphoryl group, whereas the other binds the phosphoryl oxygen and the oxygen atoms of the pyrophosphate leaving group. The two metal ions are 3.9 A apart. This mechanism fits both RNA and DNA polymerases. [Modified from T. A. Steitz, Nature, Lond. 391,231 (1998).]...

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 nucleophilic 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). 

Polynucleotide polymerases attract much attention - not only because of their central role in DNA metabolism, which suggests an important link to various diseases like tumor growth, defects of the immune system, stress-associated mutagenesis, or viral infections. Several polymerases are indispensable tools for molecular biotechnology, and could be even more valuable if the range of substrates accepted, or their stability and activity, could be tuned to specific requirements. 

Nucleotides are joined together under the influence of an enzyme called polynucleotide polymerase (PN-Pase) to give polynucleotides in the de novo synthesis. Unipolymers, for example (A) , (G) , (U) , and (C) , as well as random copolymers are produced by this method. Since the bonding occurs randomly, a molecular-weight distribution corresponding to that of a poly condensation can be expected. 

Although it is generally accepted that in vivo various types of polynucleotide polymerases exist (some using DNA as primer for RNA synthesis, others using RNA as primer), in vitro the activity of the enzymes is not necessarily restricted by the nature of the primer. In vitro both DNA and RNA may act as primers for certain RNA polymerases. 

They did find that these compounds behaved kinetically as competitive inhibitors of polymerization of the normal substrates e.g., guanosine 5 -diphosphate. These authors suggested that the successful completion of the polynucleotide phosphorylase reaction requires that the nucleotide be capable of assuming the anti conformation. Also, Kapuler and Reich (53) have found that both 8-bromo- and 8-oxoguanosine 5 -triphosphates are very poor substrates in the E. coli RNA polymerase reaction and are competitive inhibitors with respect to guanosine 5 -triphosphate as a substrate. 

C. Oligo- and Poly-nucleotides.—The stepwise enzymatic synthesis of internucleotide bonds has been reviewed. A number of polynucleotides containing modified bases have been synthesised " in the past year from nucleoside triphosphates with the aid of a polymerase enzyme, and the enzymatic synthesis of oligodeoxyribonucleotides using terminal deoxynucleotidyl transferase has been studied. Primer-independent polynucleotide phosphorylase from Micrococcus luteus has been attached to cellulose after the latter has been activated with cyanogen bromide. The preparation of insolubilized enzyme has enabled large quantities of synthetic polynucleotides to be made. The soluble enzyme has been used to prepare various modified polycytidylic acids. ... 

Primer Short synthetic polynucleotide chain (generally about 18-25 bases) to which new deoxyribonucleotides can be added by DNA polymerase. 

Kits and enzymes Superscript reverse transcriptase (Invitrogen), Maxiscript and Megascript in vitro transcription kits (Ambion, Austin, TX). Taq-DNA polymerase, T4-polynucleotide kinase, EcoRI and Hindlll restriction enzymes (Invitrogen). 

DNA polymerase I (E. coli) Reverse transcriptase Polynucleotide kinase Terminal transferase Exonuclease III... 

Polynucleotide phosphorylase was the first nucleic acid-synthesizing enzyme discovered (Arthur Kornberg s discovery of DNA polymerase followed soon thereafter). 

The reaction catalyzed by polynucleotide phosphorylase differs fundamentally from the polymerase activities discussed so far in that it is not template-dependent. The enzyme uses the 5 -diphosphates of ribonucleosides as substrates and cannot act on the homologous 5 -triphos-phates or on deoxyribonucleoside 5 -diphosphates. The RNA polymer formed by polynucleotide phosphorylase contains the usual 3, 5 -phosphodiester linkages, which can be hydrolyzed by ribonuclease. The reaction is readily reversible and can be pushed in the direction of breakdown of the polyribonucleotide by increasing the phosphate concentration. The probable function of this enzyme in the cell is the degradation of mRNAs to nucleoside diphosphates. 

The Chemistry of Nucleic Acid Biosynthesis Describe three properties common to the reactions catalyzed by DNA polymerase, RNA polymerase, reverse transcriptase, and RNA replicase. How is the enzyme polynucleotide phos-phorylase similar to and different from these three enzymes ... 

---