Deoxyribonucleic acid (DNA) contains all the information necessary for the development and function of all organisms. The replication or copying of cellular DNA occurs during the S or synthesis phase of the cell cycle (see Chapter 20). This is a necessary process to ensure that the instructions in DNA are faithfully passed on to the newly produced cells. In cell nuclei, DNA and protein complexes known as chromatin make up the chromosomes (see Chapter 6). Because replication can occur only on a single-stranded DNA template, the double-stranded DNA of the chromatin must first unwind. Once unwound, both strands of DNA are copied simultaneously. This process requires proteins to break open the double-stranded DNA, forming a replication fork.
The main enzyme that catalyzes the formation of new DNA strands is DNA polymerase. Faithful copying of DNA requires that the DNA polymerase recognize nucleotides on the opposite strand of the DNA and has a proofreading function to recognize and correct any mistakes that have been made. DNA that is being replicated experiences torsion, or twisting, created during the unwinding of DNA. Enzymes called topoisomerases act to reduce this torsional force. (Topoisomerases are an important pharmacological target for drug agents designed to inhibit DNA replication.) After replication is complete, the parent and the daughter strands of DNA must re-form a double-stranded structure as well as reestablish the chromatin structure. This area of eukaryotic molecular biology is one in which gaps in our knowledge exist. The steps and processes known to occur in the replication of prokaryotic (bacterial) DNA generally apply to eukaryotic DNA replication as well.
The structure of DNA was first described by James Watson and Francis Crick in 1953. DNA exists as a double helix, with about ten nucleotide pairs per helical turn. The spatial relationship between the two strands creates furrows in DNA—the major and minor grooves. Each of the two helical strands is composed of a sugar phosphate backbone with attached bases and is connected to a complementary strand by hydrogen bonding. The sugar in DNA is deoxyribose. The pairing of the nucleotide bases occurs such that adenine (A) binds with thymine (T) and guanine (G) with cytosine (C).
The order of the nucleotide bases determines the primary structure or sequence of the DNA. Overall, DNA is a high molecular weight double-stranded polymer (>108) of deoxyribonucleotides joined by covalent phosphodiester bonds. The phosphodiester bonds are bonds that form between the 3?-OH groups of the deoxyribose sugar on one nucleotide with the 5? phosphate groups on the adjacent nucleotide. The phosphodiester linkages between individual deoxynucleotides are directional in nature. The 5? phosphate group of one nucleotide is bound to the 3? hydroxyl group of the next nucleotide. The two complementary strands of DNA double helix therefore run in antiparallel directions. The 5? end of one strand is base-paired with the 3? end of the other strand. This primary structure is stabilized by two types of noncovalent interactions (Figure 7.1).
FIGURE 7.1.The covalent structure of DNA.
One type of noncovalent interaction within DNA includes hydrogen bonds that hold together the two strands of DNA within the double helical structure. Nucleotide bases on one strand form these bonds with nucleotide bases on the opposite strand. Adenine forms two hydrogen bonds with thymine, while guanine and cytosine are connected by three hydrogen bonds. This type of base pairing in the interior of the helix stabilizes the interior of the double-stranded DNA because the stacked bases repel each other due to their hydrophobic nature. The hydrogen bonds between bases can be made and broken easily, allowing DNA to undergo accurate replication and repair (Figure 7.2).
FIGURE 7.2.DNA double helix
CHARACTERISTICS OF EUKARYOTIC DNA SYNTHESIS
During the process of eukaryotic DNA replication, the enzymes known as DNA polymerases select the nucleotide that is to be added to the 3?-OH end of the growing chain and catalyze the formation of the phosphodiester bond. The substrates for DNA polymerases are the four deoxynucleoside triphosphates (dATP, dCTP, dGTP, and dTTP) and a single-stranded template DNA. There are distinct differences in the mechanism of DNA replication between prokaryotic and eukaryotic DNA. The focus here is on the eukaryotic processes and the following are some of the characteristic features of the eukaryotic DNA replication.
Semiconservative with respect to parental strand
One characteristic of eukaryotic DNA replication is that it is a semiconservative process. When DNA is replicated during the process of cell division, one parent or original strand of DNA is distributed to each daughter duplex in combination with a newly synthesized strand with an antiparallel orientation. Because genetic information on both strands is similar, at the end of the process, each of the two daughter strands has half new DNA and half old DNA; thus, the process is semiconservative (Figure 7.3).
FIGURE 7.3.DNA synthesis is semiconservative with respect to the parental strand.
Bidirectional with multiple origins of replication
Another characteristic of eukaryotic DNA replication is that it is bidirectional and starts in several different locations at once. DNA is copied at about 50 base pairs (bp) per second. For eukaryotic DNA consisting of 3 × 109 nucleotides, this process would take an extremely long time rather than the hours it actually does. This hastening of the replication process is made possible by the fact that replication begins at several sites on linear DNA and is completed by the end of S phase of the cell cycle (Chapter 20). As replication nears completion, “bubbles” of newly replicated DNA come together forming two new molecules (Figure 7.4).
FIGURE 7.4.Bidirectional with multiple origins of replication.
Primed by short stretches of RNA
A third characteristic of eukaryotic DNA replication is that it requires a short stretch of ribonucleic acid (RNA) for the initiation of the process. DNA polymerases cannot initiate synthesis of a complementary strand of DNA on a totally single-stranded template. A specific DNA polymerase–associated enzyme, called DNA primase, synthesizes short stretches of RNA that are complementary and antiparallel to the DNA template. The RNA primer is later removed. Chain elongation is carried out by DNA polymerases by the addition of deoxyribonucleotides to the 3? end of the growing chain. The sequence of nucleotides that are added is dictated by the base sequence of the template (or coding) strand with which the incoming nucleotides are paired (Figure 7.5).
FIGURE 7.5.Primed by short stretches of RNA.
Semidiscontinuous with respect to the synthesis of new DNA
An additional characteristic of eukaryotic DNA replication is that it is a semidiscontinuous process. A new strand of DNA is always synthesized in the 5? to 3? direction. Because the two strands of DNA are antiparallel, the strand being copied is read from the 3? end toward the 5? end.
All DNA polymerases function in the same manner: They “read” a parental strand 3? to 5? and synthesize a complementary antiparallel new strand 5? to 3?.
Because parental DNA has two antiparallel strands, the DNA polymerase synthesizes one strand in the 5? to 3? continuously. This strand is called the leading strand. The continuous or leading strand is the one in which 5? to 3? synthesis proceeds in the same direction as replication fork movement. The other new strand is synthesized 5? to 3?, but discontinuously, creating fragments that ligate (join) together later. This strand is called the discontinuous or lagging strand and is the one in which 5? to 3? synthesis proceeds in the direction opposite to the direction of the fork movement. The DNA synthesized on the lagging strand as short fragments (100 to 200 nucleotides) is called the Okazaki fragments. Although overall chain growth occurs at the base of the replication fork, synthesis of the lagging strand occurs discontinuously in the opposite direction but with exclusive 5?-3? polarity (Figure 7.6).
FIGURE 7.6.Semidiscontinuous with respect to the synthesis of new DNA. (Not to Scale)
PROTEINS INVOLVED IN DNA SYNTHESIS
Each step in the process of eukaryotic DNA replication requires the function of proteins (Figure 7.7). For example, it is important for the double-stranded DNA to open up at the multiple origins of replication and have proteins recognize and bind to DNA and prime it for replication. This process involves proteins to break the double-stranded DNA, keep the DNA structure open, synthesize a new strand, and, finally, put them together as one long linear DNA. Other proteins are needed to remove torsion that can occur when opening a double helix. Some of these proteins are described below.
FIGURE 7.7.Proofreading activity of some DNA polymerases.
Several DNA polymerases are involved in DNA replication and each of them possesses distinct activities (Table 7.1). The individual eukaryotic DNA polymerases have particular associated activities. They function as a complex to initiate DNA synthesis. Some DNA polymerases have 3?-5? exonuclease activity, or proofreading ability, that allows them to remove nucleotides that are not part of the double helix. The enzyme removes mismatched residues, thus performing an editing function. This activity enhances the fidelity of DNA replication by rechecking the correctness of base pairing before proceeding with polymerization (Figure 7.8). Eukaryotic polymerases do not possess 5?-3? exonuclease activity, an activity that is important for removing primers in prokaryotes. In eukaryotes, primer removal is done by another enzyme.
TABLE 7.1.Properties of Eukaryotic DNA Polymerases
FIGURE 7.8.Proteins involved in DNA synthesis.
DNA helicases are a class of motor proteins required to unwind short segments of the parental duplex DNA. These enzymes use energy generated from nucleotide (adenosine triphosphate [ATP]) hydrolysis to catalyze the strand separation and formation of the replication fork during DNA synthesis.
DNA primases initiate the synthesis of an RNA molecule essential for priming DNA synthesis on both the leading and the lagging strands. The first few nucleotides are ribonucleotides and the subsequent ones may be either ribonucleotides or deoxyribonucleotides.
Single-stranded DNA binding proteins
Single-stranded DNA binding proteins prevent premature annealing of the single-stranded DNA to double-stranded DNA. An important function of single-stranded DNA proteins during the process of DNA replication is to keep the strands protected until the complementary strands are produced (Figure 7.7).
DNA ligase is an enzyme that catalyzes the sealing of nicks (breaks) remaining in the DNA after DNA polymerase fills the gaps left by RNA primers. DNA ligase is required to create the final phosphodiester bond between the adjacent nucleotides on a strand of DNA (Figure 7.9).
FIGURE 7.9.Mechanism of action of DNA ligase.