Enzyme DNA polymerase

DNA polymerase enzymes all synthesize DNA by adding deoxynucleotides to the free 3 -OH group of an RNA or DNA primer sequence. The identity of the inserted nucleotide is deterrnined by its abiHty to base-pair with the template nucleic acid. The dependence of synthesis on a primer oligonucleotide means that synthesis of DNA proceeds only in a 5%o V direction if only one primer is available, all newly synthesized DNA sequences begin at the same point. 

DNA polymerase enzymes, 22 509-510 DNA polymerases, 22 513 20 447 DNA population, complexity of, 22 503 DNA-protein interactions, 2 7 608 DNA replication, 22 500 DNA restriction fragments, location of specific sequences to, 22 499 DNA sequence information... 

Double-stranded DNA has a unique property in that it is able to make identical copies of itself when supplied with precursors, relevant enzymes, and cofactors. In simplified terms, two strands begin to unwind and separate as the hydrogen bonds are broken. This produces single-stranded regions. Complementary deoxyribonu-cleotide triphosphates then pair with the exposed bases under the control of a DNA polymerase enzyme. 

Kits containing all the reagents required for DNA sequencing including the DNA polymerase enzyme, Ml3, vector, primers, buffers and labelled and unlabelled nucleotides are available from a number of manufacturers. All the components of these kits have been optimized for use with each other. 

A property of the DNA polymerase enzyme is that it only proceeds in one direction, that is, from the 5 hydroxyl to the 3 hydroxyl end of the strand. However, in donble-stranded DNA, the strands are orientated in opposite directions, that is, they are antiparallel (Fignre 20.15). Polymerisation of nucleotides along the strand that runs in the 5 to the 3 hydroxyl group direction, from the beginning to the end of the strand, poses no problems. However, there is a problem for polymerisation in the opposite direction. The solution to this problem is quite ingenious, if a little complicated. In this case, the polymerase catalyses polymerisation only in short stretches. 

Initially, the double-stranded DNA is heated to separate the strands. The primers are then added and the temperature lowered so that the primers anneal to the complementary sequences of each strand. In the presence of nucleoside triphosphates, the DNA polymerase enzyme will replicate a length of DNA starting from the 3 -end of a nucleotide, extending the chain towards the 5 -end (see Section 14.2.2). It will thus start chain extension from the 3 -ends of the primers and continue to the end of the DNA strands. This will lead to two double-stranded DNA... 

A high profile example of biocatalysis patenting is that of Taq polymerase, the thermostable DNA polymerase enzyme isolated from the thermophilic microorganism... 

The PCR is a three-step cyclic process that repeatedly duplicates a specific DNA sequence, contained between two oligonucleotide sequences called primers (154,155). The two primers form the ends of the sequence of DNA to be amplified and are normally referred to as the forward and reverse primers. The forward primer is complementary to the sense strand of the DNA template and is extended 5 to 3 along the DNA by DNA polymerase enzyme (Fig. 27). The reverse primer is complementary to the antisense strand of the DNA template and is normally situated 200-500 base pairs downstream from the forward primer, although much longer sequences (up to 50 kbase) can now be amplified by PCR. The process employs a thermostable DNA polymerase enzyme (such as the Taq polymerase from Thermus aqualicus BM) extracted from bacteria found in hot water sources, such as thermal pools or deep-water vents. These enzymes are not destroyed by repeated incubation at 94 °C, the temperature at which all double stranded DNA denatures or melts to its two separate strands (155). 

Fig. 27. The polymerase chain reaction. DNA amplification with a thermostable DNA polymerase enzyme.

Mechanism of Action. Acyclovir inhibits viral DNA replication by inhibiting the function of the DNA polymerase enzyme.42 This drug is taken into virus-infected cells and converted to acyclovir triphosphate by an enzyme known as viral thymidine kinase 42 The phosphorylated drug directly inhibits the function of the viral DNA polymerase, thus impairing the replication of viral genetic material. The virus also incorporates the drug into viral DNA strands, which halts further production of DNA because of the presence of a false nucleic acid.42... 

Mechanism of Action. Foscarnet works somewhat like acyclovir and ganciclovir that is, foscarnet inhibits the DNA polymerase enzyme necessary for viral DNA replication. Foscarnet differs from these other antiviral drugs, however, in that it does not require phosphorylation (activation) by enzymes such as viral thymidine kinase. Certain strains of viruses are thymidine-kinase deficient, meaning that these viruses lack the enzyme needed to activate antiviral agents... 

PCR works by the synthesis of two short oligonucleotides that bind correctly to opposite strands of the DNA to be replicated. This allows the DNA polymerase enzyme to begin to assemble copies of the two strands, resulting in two new DNA strands. Heating the sample causes the unwinding of the resulting double helices and provides four fresh strands that can be used as the templates for the formation of four more strands. The procedure is repeated 20-60 times over the course of a few hours either... 

Fig. 1. Comparison of enzyme-linked immuno sorbent assay (ELISA, left) and immuno-polymerase chain reaction (IPCR, right). During ELISA, an antibody-enzyme conjugate is bound to the target antigen. The enzyme converts a substrate in solution to a detectable product. In IPCR, the antibody-enzyme conjugate is replaced by an antibody-DNA conjugate. The subsequent addition of a DNA polymerase enzyme (e.g., Taq), nucleotides and a specific primer pair uses the antibody-linked DNA marker sequence as a template for amplification of the DNA. The PCR product is finally detected as an indicator of the initial amount of antigen.

Gold (ABI-Perkin Elmer, Foster City, CA). Too much DNA can lead to PCR artifacts and hence should be avoided. The four deoxynucleotide triphosphates (dNTP) include dATP, dCTP, dGTP, and dTTP. The mixture can be prepared and stored in aliquots at — 80°C. The commercial vendors that sell the Taq DNA polymerase enzyme also provide PCR reaction buffer either with or without Mg2+. The basic constituents of PCR buffer include 100 mM Tris-Cl, pH 8.3 (at room temperature), 500 mM KC1, and other additives in some brands. 

Various thermostable DNA polymerases are available commercially from various vendors. The first thermostable DNA polymerase enzyme that became available commercially was Taq DNA polymerase, isolated from T. aquaticus. This enzyme lacks 3 -to-5 proofreading exonuclease activity and hence has a higher error rate than those enzymes that possess this proofreading activity, such as pfu enzyme. For most routine purposes any thermostable DNA polymerase should suffice, irrespective of its error rate during PCR. 

When DNA undergoes replication (in preparation for cell division), an enzyme uncoils part of the double strand. Individual nucleotides naturally hydrogen bond to their complements on the uncoiled part of the original strand, and a DNA polymerase enzyme couples the nucleotides to form a new strand. This process is depicted schematically in... 

Replication of the double strand of DNA. A new strand is assembled on each of the original strands, with the DNA polymerase enzyme forming the phosphate ester bonds of the backbone. 

Newer heat-stable DNA polymerase enzymes have become available. 

In order to determine the relationship between protein structure and function and to create mutant enzymes with altered properties useful for biotechnology and cancer therapy, a directed evolution approach has been explored and novel proteins developed for Pol I DNA polymerase enzymes thymidylate synthase, thymidine kinase and 06-alkylguanine-DNA alkyltransferase. In every case the creation of a large variety of altered proteins has been achieved, and the emerging picture is that even highly conserved proteins can tolerate wide-spread amino acid changes at the active site with-... 

Random oligonucleotide mutagenesis was first applied to promotor sequences that regulate the production of enzymes in cells [21] and was the first method used to alter systematically the functions of enzymes by directed evolution [22], Based on our experience, we will focus on this approach and emphasize recent applications of this methodology to enzymes involved in DNA repair and synthesis, including DNA polymerase enzymes, thymidylate synthase, thymidine kinase, and 06-alkylguanine-DNA alkyltransferase. 

The DNA polymerase enzyme can now extend the primers and complete the replication of the rest of the DNA. The enzyme used in PCR is derived from the thermophilic bacterium Thermus aquaticus, which grows naturally in hot springs at a temperature of 90 °C, so is not denatured by the high temperatures in step 2. Its optimum temperature is about 72 °C, so the mixture is heated to this temperature for a few minutes to allow replication to take place as quickly as possible. 

The three-dimensional structures of a number of DNA polymerase enzymes are known. The first such structure to be determined was that of the so-called Klenow fragment of DNA polymerase I from E. coli (Figure 27.11). This fragment comprises two main parts of the full enzyme, including the polymerase unit. This unit approximates the shape of a right hand with domains that are referred to as the fingers, the thumb, and the palm. In addition to the polymerase, the Klenow... 

The above methods require one of the DNA polymerase enzymes and labeled dNTPs both are available commercially or can be prepared in-house. In any case, the costs of making labeled DNA sample are higher... 

The Illumina sequencing platform, which currently is the most widely used NGS platform, was commercialized in 2006, with Illumina acquiring Solexa in early 2007 (34). It is based on sequencing-by-synthesis chemistry, with reversible terminator nucleotides for the four bases, each labeled with a different fluorescent dye, and a DNA polymerase enzyme to incorporate them (35). This reaction mixture for the sequencing reactions is supplied onto the polonies created on a surface by solid-phase amplification. After each terminator nucleotide is incorporated into the DNA strand, a fluorescent signal is... 

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