DNA polymerase nucleotides

PCR requires a thermostable DNA polymerase, nucleotides of each base (collectively referred to as dNTPs)y the target sequence, and a pair of oligonucleotides (referred to as primers) complementary to opposite strands flanking the sequence to be detected. In the first step, target duplexes are denatured into single strands by heat (Figure 37-2). When the mixture is cooled, primers provided in great excess (usually over a miUion times the concentration of the initial... 

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. 

Fig. 6. DNA sequence analysis, (a) Simplified methodology for dideoxy sequencing. A primer, 5 -TCTA, hybridized to the template, is used to initiate synthesis by DNA polymerase, (b) Stmcture of 2, 3 -dideoxy CTP. When no 3 -OH functionaUty is available to support addition of another nucleotide to the growing chain, synthesis terminates once this residue is incorporated into the synthetic reaction, (c) Representation of a DNA sequencing gel and the sequence, read from bottom to the top of the gel, gives sequence information in the conventional 5 to 3 direction.

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. 

FIGURE 12.1 DNA replication yields two daughter DNA duplexes identical to the parental DNA molecule. Each original strand of the double helix serves as a template, and the sequence of nucleotides in each of these strands is copied to form a new complementary strand by the enzyme DNA polymerase. By this process, biosynthesis yields two daughter DNA duplexes from the parental double helix. 

FIGURE 12.3 The chain termination or dideoxy method of DNA sequencing, (a) DNA polymerase reaction, (b) Structure of dideoxynucleotide. (c) Four reaction mixtures with nucleoside triphosphates plus one dideoxynucleoside triphosphate, (d) Electro-phoretogram. Note that the nucleotide sequence as read from the bottom to the top of the gel is the order of nucleotide addition carried out by DNA polymerase. 

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... 

In mammahan cells, the polymerase is capable of polymerizing about 100 nucleotides per second, a rate at least tenfold slower than the rate of polymerization of deoxynucleotides by the bacterial DNA polymerase complex. This reduced rate may result from interference by nucleosomes. It is not known how the rephcation complex negotiates nucleosomes. 

Each strand of the double helix is replicated simultaneously but by somewhat different mechanisms. A complex of proteins, including DNA polymerase, replicates the leading strand continuously in the 5 to 3 direction. The lagging strand is replicated discon-tinuously, in short pieces of 150-250 nucleotides, in the 3 to 5 direction. 

Antipyrimidine antimetabolite inhibits DNA polymerase with inhibition of DNA strand elongation and replication activated in tumor cells in triphosphate form competes with conversion of cytidine to deoxycytidine nucleotides, further blocking polymerization of DNA leads to production of short DNS strands cell-cycle specific (S phase) acts only on proliferating cells. 

Enzymatic techniques can employ a variety of DNA or RNA polymerases to add controlled amounts of modified nucleotides to an existing stand. However, the most common procedures utilize either DNA polymerase I or terminal deoxynucleotide transferase. The polymerase is used with a template to add modified nucleoside triphosphates to the end of a DNA molecule or to various sites within the middle of a sequence. The terminal transferase can add modified monomers to the 3 end of a chain without a template. 

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