Chapter 5:  5.3 DNA Is Replicated by Polymerases that Take Instructions from Templates

5.3.1. DNA Polymerase Catalyzes Phosphodiester-Bond Formation

DNA polymerases catalyze the step-by-step addition of deoxyribonucleotide units to a DNA chain (Figure 5.21). Importantly, the new DNA chain is assembled directly on a preexisting DNA template. The reaction catalyzed, in its simplest form, is: where dNTP stands for any deoxyribonucleotide and PP_i is a pyrophosphate molecule. The template can be a single strand of DNA or a double strand with one of the chains broken at one or more sites. If single stranded, the template DNA must be bound to a primer strand having a free 3′-hydroxyl group. The reaction also requires all four activated precursors—that is, the deoxynucleoside 5′-triphosphates dATP, dGTP, dTTP, and dCTP—as well as Mg²⁺ ion.

The chain-elongation reaction catalyzed by DNA polymerases is a nucleophilic attack by the 3′-hydroxyl group of the primer on the innermost phosphorus atom of the deoxynucleoside triphosphate (Figure 5.22). A phosphodiester bridge forms with the concomitant release of pyrophosphate. The subsequent hydrolysis of pyrophosphate by pyrophosphatase, a ubiquitous enzyme, helps drive the polymerization forward. Elongation of the DNA chain proceeds in the 5′-to-3′ direction.

DNA polymerases catalyze the formation of a phosphodiester bond efficiently only if the base on the incoming nucleoside triphosphate is complementary to the base on the template strand. Thus, DNA polymerase is a template-directed enzyme that synthesizes a product with a base sequence complementary to that of the template. Many DNA polymerases also have a separate nuclease activity that allows them to correct mistakes in DNA by using a different reaction to remove mismatched nucleotides. These properties of DNA polymerases contribute to the remarkably high fidelity of DNA replication, which has an error rate of less than 10^-8 per base pair.

5.3.2. The Genes of Some Viruses Are Made of RNA

Genes in all cellular organisms are made of DNA. The same is true for some viruses, but for others the genetic material is RNA. Viruses are genetic elements enclosed in protein coats that can move from one cell to another but are not capable of independent growth. One well-studied example of an RNA virus is the tobacco mosaic virus, which infects the leaves of tobacco plants. This virus consists of a single strand of RNA (6930 nucleotides) surrounded by a protein coat of 2130 identical subunits. An RNA-directed RNA polymerase catalyzes the replication of this viral RNA.

Another important class of RNA virus comprises the retroviruses, so called because the genetic information flows from RNA to DNA rather than from DNA to RNA. This class includes human immunodeficiency virus 1 (HIV-1), the cause of AIDS, as well as a number of RNA viruses that produce tumors in susceptible animals. Retrovirus particles contain two copies of a single-stranded RNA molecule. On entering the cell, the RNA is copied into DNA through the action of a viral enzyme called reverse transcriptase (Figure 5.23). The resulting double-helical DNA version of the viral genome can become incorporated into the chromosomal DNA of the host and is replicated along with the normal cellular DNA. At a later time, the integrated viral genome is expressed to form viral RNA and viral proteins, which assemble into new virus particles.

Note that RNA viruses are not vestiges of the RNA world. Instead, fragments of RNA in these viruses have evolved to encode their protein coats and other structures needed for transferring from cell to cell and replicating.