Introduction to Molecular Biology: From Cells to Atoms - Anthony Rees, Michael Sternberg 2002

Nucleic Acids and Genes
Fundamentals of Replication

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Fig. 20.1.

METABOLISM/36.html">DNA Replication is the process by which the information encoded in The base sequence of a parent DNA molecule is transmitted to daughter DNA with maximum fidelity. Here we will consider the replication of double-stranded DNA only; the replication of single-stranded viral DNA is described in Chapter 4.

In semiconservative replication, first-generation daughter Cells inherit one parental DNA strand, while the second strand is newly synthesized. The same process repeats when second-generation daughter cells are formed from the first generation. Thus, only two of the four second-generation daughter cells contain a single strand of the original parental DNA, forming a double helix with a newly synthesized strand. The remaining two second-generation daughter cells contain no original parental DNA. The semiconservative replication process over two generations is illustrated in Fig. 20.1.

Meselson and Stahl demonstrated in 1958 that DNA replication proceeds via a semiconservative mechanism. They initially grew Bacteria for an extended period in a medium containing a heavy nitrogen isotope (¹⁵N), which was incorporated into the DNA, and then transferred them to a medium containing the common (light) nitrogen isotope (¹⁴N). Following replication, the first-generation daughter DNA was fractionated by density. It turned out that all daughter DNA was homogeneous and possessed a density intermediate between that of heavy and light DNA. Consequently, one strand of the daughter DNA molecule contained ¹⁵N and the other contained ¹⁴N, which corresponds to the semiconservative mechanism of replication. Base pairing in the parental DNA means that C is always positioned opposite G in one strand, and T is always opposite A. During replication, the parental DNA strands unwind, and each serves as a template that determines the base sequence in a new, complementary DNA strand. Thus, the information contained in the DNA base sequence is faithfully transmitted to the next generation.

Fig. 20.2.

Initiation of synthesis in small eukaryotic (viral) and prokaryotic (e.g., E. coli) genomes occurs at a single, strictly defined site. DNA Replication in Prokaryotes is quite rapid: in E. coli, its rate is 1700 Base Pairs per second, so that the entire genome is copied in 40 minutes. In larger cells, such as animal cells, replication is slower—proceeding at a rate of approximately 50 base pairs per second. However, initiation in this case occurs simultaneously at multiple sites along the DNA molecule; that is, many units of replication, called replicons, are formed in eukaryotic DNA. As a result, a Drosophila chromosome, for instance, consisting of 65 ∙ 10⁶ base pairs, is replicated within a few minutes. To initiate replication, double-stranded DNA must unwind at THE ORIGIN OF replication to form a loop (Fig. 20.2). A Replication fork is the segment of the DNA molecule that has already unwound and currently serves as a template for daughter DNA Synthesis. As replication proceeds, the replication fork moves along the molecule.

Bidirectional Replication is the most common mechanism of DNA replication. Following initiation, replication proceeds simultaneously in both directions along the DNA strand. To accomplish synthesis, the parent DNA double helix must unwind and its strands must separate. Various Proteins participate in these processes (Chapter 21). In linear DNA, unwinding occurs by the rotation of one strand around the other. In double-stranded circular DNA, such as E. coli DNA, unwinding and replication lead to The formation of a Structure resembling a ring with an internal loop. It is called a theta loop because its shape resembles the Greek letter Θ.

Cairns was the first to observe such loops using autoradiography. E. coli cultures were grown in a medium containing radioactive NUCLEOTIDES, which were incorporated into the DNA. The replicating DNA was then applied to photographic film, yielding an image of the Θ-loop.

Unidirectional replication is a rarer phenomenon; it is observed in certain single-stranded DNA Viruses, such as phage φX174. Unidirectional replication occurs after the formation of a double-stranded DNA form within the host Cell. The mechanism by which this is accomplished is called the rolling-circle mechanism. A break is introduced into one of the DNA strands (the outer strand in Fig. 20.2), and synthesis of a new strand begins at the 3'-end of this nicked parental strand, using the second (inner) strand as a template. This leads to the Displacement of the 5'-end of the outer strand, which subsequently serves as a template for the synthesis of another new strand.