O

Original strand

New strand

Original double-stranded DNA

Original strand

New strand

Replication is semiconservative; each of the two molecules ultimately generated contains one of the original strands (the template strand) and one newly synthesized strand.

Replication of chromosomal DNA is bidirectional; as replication proceeds in both directions, an ever-expanding "bubble" of two identical, replicated portions is created.

Replication is semiconservative; each of the two molecules ultimately generated contains one of the original strands (the template strand) and one newly synthesized strand.

Original strand

New strand

Original strand

New strand

Replication fork

Figure 7.4 Replication of Chromosomal DNA of Prokaryotes

TAC Direction of synthesis be replicated in half the time it would take if replication were unidirectional.

Replication of double-stranded DNA is semiconserva-tive. Each of the two molecules generated contains one of the original strands (the template strand) and one newly synthesized strand. Thus, the two cells produced as a result of division each have one of the original strands of DNA paired with a new complementary strand.

The process of DNA replication requires the coordinated action of many different enzymes and other proteins (table 7.1). The most critical of these exist together as a complex called a replisome. The replisome appears to act as a fixed DNA-synthesiz-ing factory, reeling in the DNA to be replicated. DNA polymerases are enzymes in the replisome that synthesize DNA, using one strand as a template to generate the complementary strand. These enzymes can only add nucleotides onto a preexisting fragment of nucleic acid, either DNA or RNA. Thus, the fragment serves as a primer from which synthesis can continue.

DNA is synthesized one nucleotide at a time as the deoxynucleoside triphosphates (dATP, dGTP, dCTP, and dTTP) are covalently joined to the nucleotide at the 3' end of the growing strand. Hydrolysis of a phosphate bond in the incoming molecule provides energy for the reaction. DNA polymerase always

Template strand

DNA polymerase

Figure 7.5 The Process of DNA Synthesis DNA polymerase synthesizes a new strand by adding one nucleotide at a time to the 3' end of the elongating strand. Because DNA is synthesized in the 5' to 3' direction, the enzyme must "read" the template strand in the 3' to 5' direction.The base-pairing rules determine the specific nucleotides that are added.

elongates the chain in the 5' to 3' direction. Because the two DNA strands are antiparallel, however, the enzyme must "read'' the template strand in the 3' to 5' direction (figure 7.5). The base-pairing rules determine the specific nucleotides added.

The replication process is very accurate, resulting in only one mistake approximately every billion nucleotides. Part of the reason for this remarkable precision is the proofreading ability of some DNA polymerases. If an incorrect nucleotide is incorporated into the growing chain, the enzyme can edit the mistake by replacing that nucleotide before moving on.

Table 7.1 Components of DNA Replication in Prokaryotes

Component

Comments

Primer

Fragment of nucleic acid to which DNA polymerase can add nucleotides (the enzyme can only add nucleotides to a preexisting fragment).

DNA gyrase

Enzyme that temporarily breaks the strands of DNA, relieving the tension caused by unwinding the two strands of the DNA helix; it is the target of a class of antibacterial medications called fluoroquinolones.

DNA ligase

Enzyme that joins two DNA fragments by forming a covalent bond between the sugar-phosphate residues of adjacent nucleotides.

DNA polymerases

Enzymes that synthesize DNA; they use one strand of DNA as a template to generate the complementary strand. Synthesis always occurs in the 5' to 3' direction.

Helicases

Enzymes that unwind the DNA helix ahead of the replication fork.

Okazaki fragment

Nucleic acid fragment generated during discontinuous replication of the lagging strand of DNA.

Origin of replication

Distinct region of a DNA molecule at which replication is initiated.

Primase

Enzyme that synthesizes small fragments of RNA to serve as primers for DNA synthesis during discontinuous replication of the lagging strand.

172 Chapter 7 The Blueprint of Life, from DNA to Protein

It takes approximately 40 minutes for the chromosome of E. coli to be replicated, regardless of the environmental conditions. How, then, can E. coli sometimes multiply with a generation time of only 20 minutes? Under favorable growing conditions, a cell initiates replication before the preceding round of replication is completed. In this way, the two progeny resulting from cell division each will get one complete chromosome that has already started another round of replication. ■ generation time, p. 85

Initiation of DNA Replication

To begin the process of DNA replication, specific proteins must recognize and bind to a distinct region of the DNA, called an origin of replication. All molecules of DNA, including chromosomes and plasmids, must have this region of approximately 250 nucleotides for replication to be initiated. The binding of the proteins causes localized denaturation, or melting, of a specific region within the origin. Using the exposed single strands as templates, small fragments of RNA are synthesized to serve as primers for DNA synthesis. The enzymes that synthesize RNA do not require a primer.

The Replication Fork

The bidirectional progression of replication around a circular DNA molecule creates two advancing Y-shaped regions where active replication is occurring. Each of these is called a replication fork. The template strands continue to "unzip'' at each fork due to the activity of enzymes called helicases. Synthesis of one new strand proceeds continuously in the 5' to 3' direction, as fresh single-stranded template DNA is exposed (figure 7.6). This strand is called the leading strand. Synthesis of the opposing strand, the lagging strand, is considerably more complicated because the DNA polymerase cannot add nucleotides to the 5' end of DNA. Instead, synthesis must be reinitiated periodically as advancement of the replication fork exposes more of the template DNA. Each initiation event must be preceded by the synthesis of an RNA primer by the enzyme primase. The result is the synthesis of a series of fragments, called Okazaki fragments, each of which begins with a short stretch of RNA. As DNA polymerase adds nucleotides to the 3' end of an Okazaki fragment, it eventually reaches the initiating point of the previous fragment. A different type of DNA polymerase then removes those RNA primer nucleotides and simultaneously replaces them with deoxynucleotides. The enzyme DNA ligase seals the gaps between fragments by catalyzing the formation of a covalent bond between the adjacent nucleotides.

Several other proteins are also involved in DNA replication. Among them is DNA gyrase, an enzyme that temporarily breaks the strands of DNA, relieving the tension caused by the unwinding of the two strands of the DNA helix. This enzyme is one of the targets of ciprofloxacin and other members of a class of antibacterial drugs called fluoroquinolones. By inhibiting the function of gyrase, the fluoroquinolones interfere with bacterial DNA replication and prevent the growth of bacteria. ■ fluoroquinolones, p. 517

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