DNA polymerase, function

Fig. 4.3.1. Schemes for genetic selection of DNA polymerase function based on complementation, (a) Host cells of E. coli recA718po/A12 that encodes a temperature-sensitive variant of DNA polymerase I (Pollts) are transformed with a polymerase mutant library. Active polymerase mutants substitute for DNA polymerase I at the non-permissive temperature (37 °C). (b) The host strain E. coli...

While the replacement DNA synthesis method for 3 -end labeling requires both the 3 - 5 exonuclease and DNA polymerase functions of DNA polymerases, the activity of the 5 - 3 DNA polymerase alone is sufficient to produce radiolabeled blunt-ended DNA frj ents with substrates containing protruding 5 termini. For example, the recessed 3 terminus shown in Fig. 1C can be extended by DNA polymerase in the presence of dXTP and dCTP to yield the blunt-ended DNA fragment indicated. 

Fig. 2. Strategies for uniform labeling of double-stranded DNA. (A) Nick translauon involves the 5 - 3 exonuclease and DNA polymerase functions of E. coli DNA polymerase 1 in the translocation of a single-strand break in a DNA strand. Trcuislocation of the breakpoint occurs in the 5 - 3 direction as a result of concomitant nucleotide hydrolysis and polymerization. (B) Template switching involves the extension of a DNA chain at a single-strand break, in a reaction where DNA is duplicated, rather than replaced as in nick treuislation.

An unusual feature of the retrovirus family, of which HIV is a member, is the RT enzyme. This enzyme is essential for replication and has the capacity to generate DNA from RNA. The RNA-dependent DNA polymerase function of the RT provides at least one target for drug discovery. Nucleoside analogs once again proved to be effective inhibitors. Wellcome s experience in the nucleo-... 

C(4 )-modified nucleotides as chemical tools for the investigation and modulation of DNA polymerase function 04SL217. 

Probing DNA polymerase function with synthetic nucleotides 04S1. 

Getting a grip on how DNA polymerases function. Nat. Struct. Biol., 8, 656-659. 

Kunkel, T. A. (2003). Considering the cancer consequences of altered DNA polymerase function. Cancer Cell 3, 105-110. 

Second important DNA polymerases function in living systems is participation in the DNA repair, aimed at correcting errors of the DNA synthesis during replication as well as numerous injuries that occur in DNA as a result of chemical and physical factors [2], Most of the reparation process involves removing the damaged fragment with subsequent single-stranded DNA synthesis that is performed by DNA polymerases. 

DNA polymerases function only in the 5 to 3 direction (Weiss-bach, 1977), suggesting that the discontinuous synthesis of DNA must occur on at least one strand of the DNA double helix. The discontinuous fragments (Okazaki fragments) (Okazaki and Okazaki, 1969) are joined by the action of ligases to form replicon-sized pieces, which polymerize... 

Determination of DNA Sequence Information. Almost all DNA sequence is determined by enzymatic methods (12) which exploit the properties of the enzyme DNA polymerase. Whereas a chemical method for DNA sequencing exists, its use has been supplanted for the most part in the initial deterrnination of a sequence. Chemical or Maxam-Gilbett sequencing (13) is mote often used for mapping functional sites on DNA fragments of known sequence. 

Another class of DNA-binding proteins are the polymerases. These have a nonspecific interaction with DNA because the same protein acts on all DNA sequences. DNA polymerase performs the dual function of DNA repHcation, in which nucleotides are added to a growing strand of DNA, and acts as a nuclease to remove mismatched nucleotides. The domain that performs the nuclease activity has an a/P-stmcture, a deep cleft that can accommodate double-stranded DNA, and a positively charged surface complementary to the phosphate groups of DNA. The smaller domain contains the exonuclease active site at a smaller cleft on the surface which can accommodate a single nucleotide. 

A number of different DNA polymerase molecules engage in DNA replication. These share three important properties (1) chain elongation, (2) processivity, and (3) proofreading. Chain elongation accounts for the rate (in nucleotides per second) at which polymerization occurs. Processivity is an expression of the number of nucleotides added to the nascent chain before the polymerase disengages from the template. The proofreading function identifies copying errors and corrects them. In E coli, polymerase III (pol III) functions at the... 

DNA has two broad functions replication and expression. First, DNA must be able to replicate itself so that the information coded into its primary structure is transmitted faithfully to progeny cells. Second, this information must be expressed in some useful way. The method for this expression is through RNA intermediaries, which in turn act as templates for the synthesis of every protein in the body. The relationships of DNA to RNA and to protein are often expressed in a graphic syllogism called the central dogma. The concept was proposed by Crick in 1958 and was revised in 1970 to accommodate the discovery of the RNA-dependent DNA polymerase. Crick s original theory suggested that the flow of information was always from RNA to protein and could not be reversed, yet it allowed for the possibility of DNA synthesis from RNA. 

DNA polymerase I is a nonessential enzyme, since viable E. coli mutants lack it (pol A). This conclusion is complicated, however, since the enzyme catalyzes three separate chemical reactions. It polymerizes deoxyribonucleoside triphosphates, and it has two exonucleolytic activities, a 3 to 5 activity and a 5 to 3 activity. The pol A - mutants lack only the polymerization activity. Other mutants lacking both the polymerase and the 5 to 3 exonuclease activity are lethal. Thus the exonuclease function is the more important one. This fits with the role of this enzyme in removing damaged DNA segments (DNA repair) and in removing covalently attached RNA from DNA chains. We will later see that small RNAs serve as primers of DNA synthesis. 

DNA polymerases from several different animal cells have been isolated and studied. The three DNA polymerases of animal cells, called a, p, and y, can be distinguished by their molecular weights, template specificity, and sensitivity to sulfhydryl reagents. Table 14.3 compares the three in regard to these differences. DNA polymerase a is probably the most important for DNA replication. This enzyme shares many functional properties with DNA polymerase III of E. coli ... 

There s not just one DNA polymerase there s a whole army. DNA replication actually occurs in large complexes containing many proteins and sometimes many polymerases. In eukaryotic cells we have to replicate both mitochondrial and nuclear DNA, and there are specific DNA polymerases for each. In addition to DNA replication, you have to make new DNA when you repair. Consequently, the function may be specialized for repair or replication. There can also be specialization for making the leading or lagging strand. Some of the activities of DNA polymerases from eukaryotes and prokaryotes are shown in the table on the next page. 

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