BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

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

24.19. One DNA Strand Is Synthesized Discontinuously

Let us return to the molecular interactions during Replication. At the Replication fork, both strands of the parental DNA serve as templates for the synthesis of new DNA. Recall that the parental DNA strands are antiparallel. Consequently, the overall direction of DNA Synthesis must be 5' → 3' for one of the daughter strands and 3' → 5' for the other (Fig. 24.39). However, all known DNA polymerases synthesize DNA in the 5' → 3' direction, not 3' → 5'. How, then, does one of the daughter strands appear (at low method resolution) to grow in the 3' → 5' direction?

Class="center">Fig. 24.39. At low resolution, the apparent direction of METABOLISM/36.html">DNA replication appears to be 5' → 3' for one daughter strand and 3' → 5' for the other. In reality, both strands are synthesized in the 5' → 3' direction, as shown in Fig. 24.40

This problem was solved by Reiji Okazaki, who discovered that a significant portion of newly synthesized DNA exists as short fragments. These fragments, about 1000 NUCLEOTIDES long (known as Okazaki fragments), persist briefly in the immediate vicinity of the replication fork. As replication proceeds, these fragments are covalently joined to each other by DNA ligase to form one of the daughter strands (Fig. 24.40). The other new strand is synthesized continuously or nearly continuously. The strand formed from Okazaki fragments is called the lagging strand, whereas the one synthesized without breaks or with very few breaks is called the leading strand. Both Okazaki fragments and the leading strand are synthesized in the 5' → 3' direction. The discontinuous assembly of the lagging strand allows overall chain growth in the 3' → 5' direction to be achieved at the atomic level through 5' → 3' polymerization.

Fig. 24.40. Schematic representation of the replication fork. Both DNA strands are synthesized in the 5' → 3' direction. The leading strand is synthesized continuously, whereas the lagging strand is synthesized as short fragments (Okazaki fragments)

24.20. RNA Serves as a Primer for DNA Synthesis

How does DNA synthesis begin? Recall that all DNA polymerases require a primer with a free 3'-OH group to initiate DNA synthesis. What serves as the primer for the Synthesis of the leading strand and Okazaki fragments? An important clue to solving this question came from the observation that RNA Synthesis is required to initiate DNA synthesis. Based on this discovery, it was hypothesized that RNA evidently serves as a primer in DNA synthesis, since it was already known that RNA polymerases are capable of initiating chain synthesis de novo. It was subsequently shown that the newly formed DNA is covalently linked to a short RNA fragment, which acts as the primer. Thus, An RNA primer is involved in DNA synthesis.

In all probability, DNA replication in E. coli Cells proceeds as shown in Fig. 24.41.

Fig. 24.41. Initiation of DNA synthesis. A - primase synthesizes a short complementary RNA strand; B - this RNA serves as a primer for the synthesis of new DNA; C - the RNA incorporated into the newly formed strand is hydrolyzed, creating a gap that is subsequently filled

1. A specialized RNA polymerase (called primase) synthesizes a short RNA chain (approximately 10 nucleotides long) complementary to one of the template DNA strands. Unlike DNA polymerase, primase does not require a primer to synthesize a polynucleotide.

2. The 3'-hydroxyl group of the terminal ribonucleotide of this RNA chain serves as a primer for DNA synthesis catalyzed by the DNA polymerase III holoenzyme. Most of the newly formed DNA is synthesized by this multi-subunit complex.

3. The RNA component of this RNA-DNA hybrid is hydrolyzed by DNA polymerase I.

4. Following the removal of RNA from the newly formed strands, fairly large gaps remain between the DNA fragments. DNA polymerase I, which is well adapted

for DNA synthesis on a single-stranded template, fills in these gaps.

Recent studies have shown that the action of primase is preceded by The formation of a prepriming intermediate complex consisting of at least five Proteins. One of these—the dnaB protein—can move along the DNA using the energy of ATP Hydrolysis. The dnaB protein can serve as a signal to activate primase. The Specificity of initiating each replication cycle may be ensured by proteins that deliver the dnaB protein precisely to the region of the ilv Gene, where THE ORIGIN OF replication of the E. coli chromosome is located. The timing of DNA replication initiation is of critical importance because it must be coordinated with Cell Division. Indeed, the bacterial chromosome is associated with an invagination of The Cell membrane (Fig. 24.42).

Fig. 24.42. Electron micrograph of an E. coli chromosome attached to two fragments of the cell membrane. The image shows a single intact supercoiled DNA molecule

24.21. The Energy of ATP Hydrolysis Is Used to Unwind Parental DNA at the Replication Fork by the rep Protein

In 1953, Watson and Crick noted that "unwinding The Double Helix is a formidable task." Recent studies have shown that in the E. coli cell, the parental double helix is actively unwound at the replication fork through the action of an enzyme known as the rep protein. Because the energy required to unwind the parental DNA is released during ATP hydrolysis, the rep protein is referred to as a helicase. Approximately two ATP molecules are consumed for every base pair separated. Subsequently, each separated strand of parental DNA interacts with several molecules of a single-stranded DNA-binding protein (SSB protein). The Role of the SSB protein is to stabilize the single-stranded DNA regions generated by the helicase action, enabling the unwound region to function as a template. SSB proteins are also known as helix-destabilizing proteins (HD proteins) or melting proteins.

24.22. DNA gyrase introduces negative supercoils into the parental DNA to facilitate its unwinding

The unwinding of a covalently closed circular DNA molecule creates topological problems, as unwinding the double helix induces positive supercoiling in the closed molecule. At the replication fork, the parental DNA rotates at a rate of 100 rpm — more than 100 times faster than a standard long-playing vinyl record. For the unwinding process to continue, these rotation-induced positive supercoils must be removed. In other words, some form of molecular swivel is required. Martin Gellert recently discovered that this function is performed by DNA gyrase. This topoisomerase removes positive supercoils by introducing single-strand breaks and subsequently resealing the phosphodiester bonds in the DNA backbone. ATP is not required for this thermodynamically favorable relaxation of the tertiary Introduction/20.html">DNA Structure. Furthermore, DNA gyrase can actively introduce negative supercoils into covalently closed circular DNA at the expense of ATP hydrolysis energy (Fig. 24.43). These negative supercoils facilitate the Separation of the parental DNA strands at the replication fork¹.

¹ In this chapter, the author uses the term DNA gyrase to refer to two entirely different Enzymes. One of them is DNA gyrase, which is capable of introducing thermodynamically unfavorable negative supercoils into DNA utilizing the energy of ATP hydrolysis; the other is an ATP-independent DNA topoisomerase that brings a circular DNA molecule into a thermodynamically equilibrium (relaxed) state. These are completely distinct proteins with different inhibitors, ionic environment requirements, and Mechanisms of action. DNA gyrase Functions as the molecular swivel during replication. — Trans. Note.

Fig. 24.43. Catalytic activities of DNA gyrase