DNA Replication:- DNA Is Synthesized by DNA Polymerases
The search for an enzyme that could synthesize DNA began in 1955. Work by Arthur Kornberg and colleagues led to the purification and characterization of DNA polymerase from E. coli cells, a single-polypeptide en zyme now called DNA polymerase I(Mr103,000; encoded by the polA gene). Much later, investigators found that E. coli contains at least four other distinct DNA polymerases, described below. Detailed studies of DNA polymerase I revealed features of the DNA synthetic process that are now known to be common to all DNA polymerases. The fundamental reaction is a phosphoryl group transfer. The nucleophile is the 3-hydroxyl group of the nucleotide at the 3 end of the growing strand. Nucleophilic attack occurs at the α phosphorus of the incoming deoxynucleoside 5-triphosphate (Fig. 1). Inorganic pyrophosphate is released in the reaction. The general reaction is
where dNMP and dNTP are deoxynucleoside 5-mono phosphate and 5-triphosphate, respectively. The reaction appears to proceed with only a minimal change in free energy, given that one phosphodiester bond is formed at the expense of a somewhat less stable phosphate anhydride. However, noncovalent base-stacking and base-pairing interactions provide additional stabilization to the lengthened DNA product relative to the free nucleotide. Also, the formation of products is facilitated in the cell by the 19 kJ/mol generated in the sub sequent hydrolysis of the pyrophosphate product by the enzyme pyrophosphatase.
MECHANISM FIGURE 1 Elongation of a DNA chain. (a) DNA polymerase I activity requires a single unpaired strand to act as template and a primer strand to provide a free hydroxyl group at the 3 end, to which a new nucleotide unit is added. Each incoming nucleotide is selected in part by base pairing to the appropriate nucleotide in the template strand. The reaction product has a new free 3 hydroxyl, allowing the addition of another nucleotide. (b) The catalytic mechanism likely involves two Mg+2 ions, coordinated to the phosphate groups of the incoming nucleotide triphosphate and to three Asp residues, two of which are highly conserved in all DNA polymerases. The top Mg+2 ion in the figure facilitates attack of the 3-hydroxyl group of the primer on the phosphate of the nucleotide triphosphate; the lower Mg+2 ion facilitates displacement of the pyrophosphate. Both ions stabilize the structure of the pentacovalent transition state. RNA polymerases use a similar mechanism .
Early work on DNA polymerase I led to the definition of two central requirements for DNA polymerization. First, all DNA polymerases require a template. The polymerization reaction is guided by a template DNA strand according to the base-pairing rules predicted by Watson and Crick: where a guanine is present in the template, a cytosine deoxynucleotide is added to the new strand, and so on. This was a particularly important discovery, not only because it provided a chemical basis for accurate semiconservative DNA replication but also because it represented the first example of the use of a template to guide a biosynthetic reaction. Second, the polymerases require a primer. A primer is a strand segment (complementary to the template) with a free 3-hydroxyl group to which a nucleotide can be added; the free 3 end of the primer is called the primer terminus. In other words, part of the new strand must already be in place: all DNA polymerases can only add nucleotides to a preexisting strand. Most primers are oligonucleotides of RNA rather than DNA, and specialized enzymes synthesize primers when and where they are required. After adding a nucleotide to a growing DNA strand, a DNA polymerase either dissociates or moves along the template and adds another nucleotide. Dissociation and reassociation of the polymerase can limit the overall polymerization rate—the process is generally faster when a polymerase adds more nucleotides without dis sociating from the template. The average number of nucleotides added before a polymerase dissociates defines its processivity. DNA polymerases vary greatly in processivity; some add just a few nucleotides before dissociating, others add many thousands.
