PART 1 Introduction
4.3. DNA REPLICATION: PRESERVING AND PROPAGATING THE CELLULAR MESSAGE
The double-helix structure of DNA discussed in Chapter 2 is extremely well suited to its role of preserving genetic information. Information resides simply in the linear arrange- ment of the four nucleotide letters (A, T, G, and C). Because G can hydrogen-bind only to C and A only to T, the strands must be complementaryif an undistorted double helix is to result. Replication is semiconservative (see Fig. 2.18); each daughter chromosome con- tains one parental strand and one newly generated strand.
To illustrate the replication process (see Figs. 4.2 and 4.3), let us briefly consider DNA replication in E. coli. The enzyme responsible for covalently linking the monomers is DNA polymerase. Escherichia colihas three DNA polymerases (named Pol I, Pol II, and Pol III). A DNA polymerase is an enzyme that will link deoxynucleotides together to form a DNA polymer. Pol III enzymatically mediates the addition of nucleotides to an RNA primer. Pol I can hydrolyze an RNA primer and duplicates single-stranded regions of DNA; it is also active in the repair of DNA molecules. The exact role of Pol II is still unclear.
In addition to the enzyme, the enzymatic reaction requires activated monomer and the template. The activated monomers are the nucleoside triphosphates. The formation of
108 How Cells Work Chap. 4 Figure 4.2. Initiation of DNA synthesis re- quires the formation of an RNA primer.
the 5¢–3¢phosphodiester bond to link a nucleotide with the growing DNA molecule re- sults in the release of a pyrophosphate, which provides the energy for such a biosynthetic reaction. The resulting nucleoside monophosphates are the constituent monomers of the DNA molecule.
Replication of the chromosome normally begins at a predetermined site, the origin of replication, which in E. coliis attached to the plasma membrane at the start of replica- tion. Initiator proteins bind to DNA at the origin of replication, break hydrogen bonds in the local region of the origin, and force the two DNA strands apart. When DNA replica- tion begins, the two strands separate to form a Y-shaped structure called a replication fork.
Movement of the fork must be facilitated by the energy-dependent action of DNA gyrase and unwinding enzymes. In E. colithe chromosome is circular. In E. coli(but not all or- ganisms) the synthesis of DNA is bidirectional. Two forks start at the origin and move in opposite directions until they meet again, approximately 180∞from the origin.
To initiate DNA synthesis, an RNA primeris required; RNA polymerase requires no primer to initiate the chain-building process, while DNA polymerase does. (We can spec- ulate on why this is so. In DNA replication, it is critical that no mistakes be made in the addition of each nucleotide. The DNA polymerase, Pol III, can proofread, in part due to the enzyme’s 3¢-to-5¢ exonuclease activity, which can remove mismatches by moving backward. On the other hand, a mistake in RNA synthesis is not nearly so critical, so RNA polymerase lacks this proofreading capacity.) Once a short stretch of RNA comple- mentary to one of the DNA strands is made, DNA synthesis begins with Pol III. Next, the RNA portion is degraded by Pol I, and DNA is synthesized in its place. This process is summarized in Fig. 4.2.
DNA polymerase works only in the 5¢-to-3¢direction, which means that the next nu- cleotide is always added to the exposed 3¢-OH group of the chain. Thus, one strand (the leading strand) can be formed continuously if it is synthesized in the same direction as the replication fork is moving. The other strand (the lagging strand) must be synthesized dis- continuously. Short pieces of DNA attached to RNA are formed on the lagging strand.
These fragments are called Okazaki fragments. The whole process is summarized in Fig.
4.3. The enzyme, DNA ligase, which joins the two short pieces of DNA on the continuous strand, will be very important in our discussions of genetic engineering.
Figure 4.3. Schematic representation of the steps of replication of the bacterial chromo- some. Part (a) represents a portion of a replicating bacterial chromosome at a stage shortly after replication has begun at the origin. The newly polymerized strands of DNA (wavy lines) are synthesized in the 5¢-to-3¢direction (indicated by the arrows), using the preex- isting DNA strands (solid lines) as a template. The process creates two replication forks, which travel in opposite directions until they meet on the opposite side of the circular chromosome, completing the replication process. Part (b) represents a more detailed view of one of the replicating forks and shows the process by which short lengths of DNA are synthesized and eventually joined to produce a continuous new strand of DNA. For pur- poses of illustration, four short segments of nucleic acid are illustrated at various stages.
In (1), primer RNA (thickened area) is being synthesized by an RNA polymerase (R Pol).
Then, successively, in (2) DNA is being polymerized to it by DNA polymerase III (Pol III); in (3) a preceding primer RNA is being hydrolyzed, while DNA is being polymerized in its place by the exonuclease and polymerase activities of DNA polymerase I (Pol I); fi- nally, the completed short segment of DNA (4) is joined to the continuous strand (5) by the action of DNA ligase. (With permission, from R. Y. Stanier and others, The Microbial World, 5th ed., Pearson Education, Upper Saddle River, NJ, 1986, p. 133.)
This brief discussion summarizes the essentials of how one DNA molecule is made from another and thus preserves and propagates the genetic information in the original molecule. Now we turn our attention to how this genetic information can be transferred.