9.3: DNA Polymerases Catalyze Replication

Before considering what happens at replication forks in detail, let’s look at the role of DNA polymerases in the process. The first DNA polymerase enzyme was discovered in E. coli by Arthur Kornberg, for which he received the 1959 Nobel Prize in Chemistry. However, the rate of catalysis of new DNA, at least in vitro, was too slow to account for the in vivo rate of E. coli replication. It was Thomas Kornberg, one of Arthur’s sons, who later found two additional, faster-acting DNA polymerases. (We already met the older Kornberg brother, Roger!)

All DNA polymerases require a template strand against which to synthesize the new complementary strand. In successive condensation reactions, all DNA polymerases catalyze the addition of nucleotides to the 3′ end of the growing DNA strand. Finally, all DNA polymerases also have the odd property that they can only add to a preexisting strand of nucleic acid, raising the question of where the “preexisting” strand comes from! The polymerases catalyze the formation of a phosphodiester linkage between the end of a growing strand and an incoming nucleotide. The latter must complement a nucleotide in the template strand. Energy for the formation of the phosphodiester linkage comes in part from the hydrolysis of two phosphates (pyrophosphate) from the incoming nucleotide during the reaction. While replication requires the participation of many nuclear proteins in both prokaryotes and eukaryotes, polymerases perform the basic steps of replication (Figure 9.5).

Figure 9.5: DNA polymerases require a template strand and use deoxynucleotide triphosphate precursors (upper illustration) to catalyze the replication of new DNA in the 5′ to 3’ direction, shown here extending the new DNA by one nucleotide (lower illustration).

178 DNA Polymerases & Their Activities

Although DNA polymerases replicate DNA with high fidelity and make as few errors as one per \(10^7\) nucleotides, mistakes do occur. The proofreading ability of some DNA polymerases corrects many of these mistakes. The polymerase can sense a mismatched base pair, slow down, and then catalyze repeated hydrolyses of nucleotides until it reaches the mismatched base pair. Figure 9.6 illustrates this basic proofreading by a DNA polymerase.

Figure 9.6: If during replication, a wrong nucleotide is inserted in a growing DNA (upper illustration), some DNA polymerases will detect and proofread the incorrect nucleotide and replace it with the correct one (middle illustration). Replication will then resume (lower illustration.

After mismatch repair, the DNA polymerase resumes its forward movement at replication forks. Of course, not all mistakes are caught by this or other repair mechanisms (see section 9.5, “DNA Repair”). While mutations in eukaryotic germline cells that escape correction can cause genetic diseases, most of these replication errors are the very mutations that fuel evolution. Without mutations in germline cells (i.e., egg and sperm cells), there would be no mutations and no evolution, and without evolution, life itself would have reached a quick dead end! On the other hand, replication mistakes can generate mutations in somatic cells. If these somatic mutations escape correction, they can have serious consequences, including the generation of tumors and cancers.

CHALLENGE

In classical genetic terms, any mutation in a gene that produces an inactive enzyme would be recessive. In old-fashioned Mendelian genetic terms, what would the genotype and phenotype be of the progeny of a cross between a male and a female that are both heterozygous for the gene for this enzyme?