Genes VII
13.2 DNA polymerases are the enzymes that make DNA |
| Key terms defined in this section |
| DNA polymerases are enzymes that synthesize a daughter strand(s) of DNA (under direction from a DNA template). May be involved in repair or replication.DNA replicase is a DNA-synthesizing enzyme required specifically for replication.Repair of damaged DNA can take place by repair synthesis, when a strand that has been damaged is excised and replaced by the synthesis of a new stretch. It can also take place by recombination reactions, when the duplex region containing the damaged is replaced by an undamaged region from another copy of the genome.Replication of duplex DNA takes place by synthesis of two new strands that are complementary to the parental strands. The parental duplex is replaced by two identical daughter duplexes, each of which has one parental strand and one newly synthesized strand. It is called semiconservative because the conserved units are the single strands of the parental duplex. |
There are two basic types of DNA synthesis.
| Figure 13.1 Semiconservative replication synthesizes two new strands of DNA. |
Figure 13.1 shows the result of semiconservative replication. The two strands of the parental duplex are separated, and each serves as a template for synthesis of a new strand. The parental duplex is replaced with two daughter duplexes, each of which has one parental strand and one new ly synthesized strand.
| Figure 13.2 Repair synthesis replaces a damaged strand of DNA. |
Figure 13.2 shows the consequences of a repair reaction. One strand of DNA has been damaged. It is excised and new material is synthesized to replace it. (Repair synthesis is not the only way to replace damaged DNA; the reactions involved in replacement of damaged sequences are discussed in 14 Recombination and repair.)
An enzyme that can synthesize a new DNA strand on a template strand is called a DNA polymerase. Both prokaryotic and eukaryotic cells contain multiple DNA polymerase activities. Only some of these enzymes actually undertake replication; sometimes they are called DNA replicases. The others are involved in subsidiary roles in replication and/or participate in repair synthesis.
| Figure 13.3 DNA synthesis occurs by adding nucleotides to the 3 F-OH end of the growing chain, so that the new chain is synthesized in the 5 F-3 F direction. The precursor for DNA synthesis is a nucleoside triphosphate, which loses the terminal two phosphate groups in the reaction. |
All prokaryotic and eukaryotic DNA polymerases share the same fundamental type of synthetic activity. Each can extend a DNA chain by adding nucleotides one at a time to a 3′ VOH end, as illustrated diagrammatically in Figure 13.3. The choice of the nucleotide to add to the chain is dictated by base pairing with the template strand.
It is convenient to think of DNA-synthesizing activities in terms of the DNA polymerase enzymes; but it is a moot point whether a DNA replicase enzyme exists as a discrete entity. (We consider the same issue for RNA polymerase in 9 Transcription.) Bacterial DNA replicase activity is recovered as aggregates containing various "subunits." The DNA-synthesizing activity is only one of several functions associated in the replisome.
| Figure 13.4 Only one DNA polymerase is the replicase. The others participate in repair of damaged DNA. |
Figure 13.4 summarizes the DNA polymerases that have been characterized in E. coli. DNA polymerase I (coded by polA) is involved in the repair of damaged DNA and, in a subsidiary role, in semiconservative replication. DNA polymerase III, a multisubunit protein, is the replicase responsible for de novo synthesis of new strands of DNA. Other enzymes (DNA polymerases II, IV and V) are involved in specific repair reactions.
When extracts of E. coli are assayed for their ability to synthesize DNA, the predominant enzyme activity is DNA polymerase I. Its activity is so great that it makes it impossible to detect the activities of the enzymes actually responsible for DNA replication! To develop in vitro systems in which replication can be followed, extracts are therefore prepared from polA mutant cells.
Some phages code for DNA polymerases. They include T4, T5, T7, and SPO1. The enzymes all possess 5′ V3′ synthetic activities and 3′ V5′ exonuclease proofreading activities (see next section). In each case, a mutation in the gene that codes for a single phage polypeptide prevents phage development. Each phage polymerase polypeptide associates with other proteins, of either phage or host origin, to make the intact enzyme.
Several classes of eukaryotic DNA polymerases have been identified. DNA polymerase δ is the nuclear replicase; DNA polymerase α is concerned with "priming" (initiating) replication. Other DNA polymerases are involved in repairing damaged nuclear DNA (β and ε) or with mitochondrial DNA replication (γ).