BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

CHAPTER 24 DNA: THE GENETIC ROLE, STRUCTURE, AND REPLICATION

24.16. Discovery of DNA Polymerases II and III

We have seen that DNA polymerase I can synthesize and repair DNA in vitro. Does it perform these Functions in vivo? This is a very legitimate question, as an enzyme is not always able to carry out in vivo the exact reaction it catalyzes in vitro. The intracellular environment may differ significantly from the conditions used in in vitro assays, and Other Enzymes may also be present within The Cell. Indeed, *E. coli* Cells contain at least two other DNA polymerases, designated polymerases II and III, which were discovered about 15 years after DNA polymerase I. Why did it take so long? The reason is that The activity of DNA polymerases II and III was masked by the overwhelming activity of polymerase I.

Class="center">Fig. 24.32. Mechanism of the reaction catalyzed by DNA ligase

The situation changed in 1969 when Paula DeLucia and John Cairns isolated an *E. coli* mutant whose cell extracts exhibited only 0.5% to 1% of the normal DNA polymerase I activity compared to wild-type cells. This mutant (designated polA 1) grew at the same rate as the parental strain. Furthermore, many phages replicated just as well in polA 1 cells as in the parental strain. However, this mutant was much more susceptible to killing by ultraviolet irradiation. In addition, the polA 1 mutant showed increased sensitivity to the lethal effects of the chemical mutagen methyl methanesulfonate. DeLucia and Cairns concluded that DNA Replication proceeds normally in their polA 1 mutant, whereas DNA Repair is severely impaired. They hypothesized that DNA Synthesis requires a different polymerase, distinct from DNA polymerase I.

The low Background activity of polymerase I in the polA 1 mutant greatly facilitated the search for new DNA polymerases. Soon, two such enzymes were isolated and characterized in several laboratories. DNA polymerases II and III resemble polymerase I in the following respects:

1. They catalyze METABOLISM/36.html">Template-Directed DNA Synthesis from deoxyribonucleoside triphosphate precursors.

2. They require a primer with a free 3'-OH group for activity.

3. Synthesis proceeds in the 5' → 3' direction.

4. They possess 3' → 5' exonuclease activity. DNA polymerase III (but not II) also functions as a 5' → 3' nuclease.

These polymerases differ in their template preferences. For polymerases II and III, the optimal templates are double-stranded DNA molecules containing short single-stranded gaps. Polymerase I, by contrast, prefers extended single-stranded regions adjacent to double-helical segments. The maximum in vitro catalytic rates for these enzymes also differ: polymerase I incorporates 10 NUCLEOTIDES per second, whereas polymerase II incorporates 0.5, and polymerase III incorporates 150 nucleotides per second. In its physiologically active state, DNA polymerase III is associated with several other Proteins. This multisubunit complex is referred to as the DNA polymerase III holoenzyme.

What is The Role of these polymerases in vivo? As we shall discuss shortly, the multienzyme complex containing DNA polymerase III synthesizes the bulk of the newly formed DNA, whereas DNA polymerase I removes the primer and fills the resulting gaps. The Physiological Role of DNA polymerase II remains to be established. Recent biochemical and genetic studies have demonstrated that, In addition to DNA polymerases I and II and DNA ligase, more than 10 proteins are required for DNA replication in *E. coli*. Before examining the interactions of these proteins with DNA, we will first look at the overall features of replication at the whole-chromosome level.

Fig. 24.33. Location of structural genes for the three *E. coli* DNA polymerases. The Gene for DNA polymerase I is located at the polA locus, that for DNA polymerase II at the polB locus, and that for DNA polymerase III at the dnaE locus

24.17. Unwinding of Parental DNA and Synthesis of New DNA Occur at the Replication Fork

Images of DNA during replication have been obtained using autoradiography and Electron Microscopy. In autoradiography, the image is formed by the radioactive decay of a suitable isotope, such as tritium. The emitted electrons interact with the silver grains of a photographic emulsion, producing black dots upon film development. Autoradiography has a relatively low resolving power, on the order of a few hundred angstroms. However, this method has a distinct advantage: it allows visualization only of molecules that contain the label. To make DNA visible by autoradiography, it is labeled by incorporating tritium-labeled thymine or thymidine.

Autoradiographs and electron micrographs show that replicating *E. coli* DNA forms a closed circle containing an internal loop (Fig. 24.34). Molecules of this shape are called theta structures because they resemble the Greek letter Ɵ (Fig. 24.35). Theta structures demonstrate that DNA molecules retain their circular conformation during replication. The resolution of this method is not high enough to distinguish free ends; however, it is clear that there are no long stretches of single-stranded DNA in the molecule. Thus, these images rule out a replication mechanism in which the parental DNA strands unwind completely before being used as templates for new DNA synthesis. Instead, the synthesis of new DNA is coupled with the simultaneous unwinding of the parental DNA. The region where simultaneous unwinding and synthesis take place is called the Replication fork.

Fig. 24.34. Autoradiograph of replicating *E. coli* DNA

Fig. 24.35. Schematic drawing of a circular *E. coli* chromosome during replication. In this theta Structure, parental DNA is shown in blue and newly synthesized DNA in red

24.18. DNA Replication Begins at a Unique Origin and Proceeds Bidirectionally

Does DNA replication in E. coli begin at a random site on the chromosome, or is there a specific initiation site? Because DNA replication is a tightly regulated process, it seems a priori much more likely that it initiates at a defined locus. Indeed, experiments determining the relative number of various genes under conditions of rapid DNA synthesis have shown that replication in E. coli cells starts at a single, specific site on the chromosome. Consider two genes, a and b. Suppose that gene a is located near THE ORIGIN OF replication, and gene b is near the terminus. In this case, gene a will be replicated much earlier than gene b. In a rapidly growing culture, there will be approximately two copies of gene a for every copy of gene b. Conversely, if DNA replication started at a random point, the amounts of genes a and b would be equal. The relative gene dosage was determined using the Hybridization method discussed below (Section 25.5). The results of these experiments clearly demonstrated that the relative gene frequency indeed depends on its map position (Fig. 24.36). These data led to the following Conclusions.

Fig. 24.36. Relative abundances of various genes during rapid DNA synthesis in E. coli as a function of their position on the genetic map

1. Replication initiates at a strictly defined, unique site near the ilv gene, located at 74' on the standard E. coli genetic map.

2. Replication proceeds simultaneously in both directions at approximately the same rate. In other words, There are two replication forks: one moving clockwise, and the other counterclockwise.

3. The two replication forks meet near the trp marker (at 25' on the genetic map)—at a point almost diametrically opposite to the origin of replication.

Further Evidence for the bidirectional Nature of DNA replication in E. coli was obtained using autoradiography. To this end, replication was initially carried out in a medium containing tritium-labeled thymine of moderate specific radioactivity. After a few minutes of incubation, the Bacteria were transferred to a medium containing highly tritiated radioactive thymine. Samples with two different levels of radioactivity were used to produce Two Types of silver grain tracks on the autoradiographs: tracks with a low grain density corresponding to the DNA synthesized initially, and high-density tracks corresponding to the DNA synthesized later. If replication were unidirectional, one would observe tracks with a high grain density at one end and a low density at the other. However, if replication is bidirectional, the middle of each DNA trace should have a low grain density, and the ends should have a high density (Fig. 24.37). The autoradiographs provided a clear answer (Fig. 24.38). The silver grain tracks (DNA traces) in all cases exhibited a higher grain density at both ends than in the middle, thereby indicating that E. coli Chromosome replication proceeds bidirectionally.

Fig. 24.37. Expected autoradiography results for unidirectional and Bidirectional Replication when bacteria are transferred from a medium containing moderately radioactive thymine to one with highly radioactive thymine

Fig. 24.38. Autoradiogram of replicating E. coli DNA (experimental conditions are described in the legend to Fig. 24.37). The observed distribution of silver grains demonstrates that replication proceeds bidirectionally