DNA molecule must be opened up before replication can proceed. The helix is a very stable structure, and in a test tube the two strands separate only when the temperature reaches about 90 °C. In the cell, the combined actions of several proteins help to separate the two strands. Much of our knowledge of replication comes from studying E. coli, but similar systems operate in all organisms, bacteria, archaea, and eukaryotes. The proteins E. coli uses to open up the double helix during replication include DnaA, DnaB, DnaC, and single‐stranded DNA‐binding proteins.
DnaA Protein
Several copies of the protein DnaA, which is activated by a molecule of ATP, bind to four sequences of nine base pairs within the E. coli origin of replication (ori C). This causes the two strands to begin to separate (or “melt”) because the hydrogen bonds in DNA are broken near to where the DnaA protein binds. The DNA is now in the open complex formation and has been prepared for the next stage in replication, which is to open up the helix even further.
DnaB and DnaC Proteins
DnaB is a helicase. It moves along a DNA strand, breaking hydrogen bonds, and in the process unwinds the helix (Figure 4.1). Two molecules of DnaB are needed, one for each strand of DNA. One DnaB attaches to one of the template strands and moves in the 5′ to 3′ direction; the second DnaB attaches to the other strand and moves in the 3′ to 5′ direction. The unwinding of the DNA double helix by DnaB is an ATP‐dependent process. DnaB is escorted to the DNA strands by another protein, DnaC. However, having delivered DnaB to its destination, DnaC plays no further role in replication.
Single‐Stranded DNA‐Binding Proteins
As soon as DnaB unwinds the two parental strands, they are engulfed by single‐stranded DNA‐binding proteins. These proteins bind to adjacent groups of 32 nucleotides. DNA covered by single‐stranded DNA‐binding proteins is rigid, without bends or kinks. It is, therefore, an excellent template for DNA synthesis. Single‐stranded binding proteins are sometimes called helix‐destabilizing proteins.
In prokaryotes the synthesis of a new DNA molecule is catalyzed by the enzyme DNA polymerase III. Its substrates are the four deoxyribonucleoside triphosphates, dGTP, dATP, dTTP, and dCTP. DNA polymerase III catalyzes the formation of a phosphodiester bond (Figure 3.3 on page 37) between the 3′ hydroxyl of the sugar residue on the most recently added nucleotide and the 5′ phosphate of the incoming nucleotide. The elongation of the new DNA molecule takes place in the 5′ to 3′ direction (Figure 4.2a). The base sequence of a newly synthesized DNA strand is dictated by the base sequence of its parental strand. If the sequence of the template strand is 3′ CATCGA 5′, then that of the daughter strand is 5′ GTAGCT 3′. In eukaryotes, DNA replication is performed by three isoforms, DNA polymerases α, δ, and ε, but the mechanism is much the same.
DNA polymerase III can only add a nucleotide to a free 3′‐hydroxyl group and therefore synthesizes DNA in the 5′ to 3′ direction. The template strand is read in the 3′ to 5′ direction. However, the two strands of the double helix are antiparallel. They cannot be synthesized in the same direction because only one has a free 3′‐hydroxyl group, the other has a free 5′‐phosphate group. No DNA polymerase has been found that can synthesize DNA in the 3′ to 5′ direction, that is, by attaching a nucleotide to a 5′ phosphate, so the synthesis of the two daughter strands must differ. One strand, the leading strand, is synthesized continuously while the other, the lagging strand, is synthesized discontinuously. DNA polymerase III can synthesize both daughter strands, but must make the lagging strand as a series of short 5′ to 3′ sections (Figure 4.1). These fragments of DNA, called Okazaki fragments after Reiji Okazaki who discovered them in 1968, are then joined together by DNA ligase.
Medical Relevance 4.1 Inhibiting DNA Polymerase Fights Cancer
Drugs that inhibit DNA replication prevent cells dividing. Cytarabine (also known as cytosine arabinoside and arabinofuranosyl cytidine) is sold as Cytosar‐U, Tarabine PFS, Depocyt, and AraC. It inhibits eukaryote DNA polymerases α, δ, and ε, and is therefore widely used to treat cancers. It is on the World Health Organization's model list of essential medicines.
Example 4.1 The Meselson and Stahl Experiment
In 1958 Matthew Meselson and Franklin Stahl designed an ingenious experiment to test whether each strand of the double helix does indeed act as a template for the synthesis of a new strand. They grew the bacterium E. coli in a medium containing the heavy isotope 15N that could be incorporated into new DNA molecules. After several cell divisions they transferred the bacteria, now containing “heavy” DNA, to a medium containing only the lighter, normal, isotope 14N. Any newly synthesized DNA molecules would therefore be lighter than the original parent DNA molecules containing 15N. The difference in density between the heavy and light DNAs allows their separation using very high‐speed centrifugation. The results of this experiment are illustrated in the figure. DNA isolated from cells grown in the 15N medium had the highest density and migrated the furthest during centrifugation. The lightest DNA was found in cells grown in the 14N medium for two generations, whereas DNA from bacteria grown for only one generation in the lighter 14N medium had a density halfway between these two. This is exactly the pattern expected if each strand of the double helix acts as a template for the synthesis of a new strand. The two heavy parental strands separated during replication, with each acting as a template for a newly synthesized light strand, which remained bound to the heavy strand in a double helix. The resulting DNA was therefore of intermediate density. Only in the second round of DNA replication, when the light strands created during the first round of replication were allowed to act as templates for the construction of complementary light strands, did DNA double helices composed entirely of 14N‐containing building blocks appear.
DNA Synthesis Requires an RNA Primer
DNA polymerase III cannot itself initiate the synthesis of DNA. The enzyme primase is needed to catalyze the formation of a short stretch of RNA complementary in sequence to the DNA template strand (