DNA Replication: Basics

Problems of Replication:
       The replication process needs that each double-helical molecule of DNA produce two identical molecules of DNA. This means that wherever a G-C or A-T base pair occurs in the parental molecule, the identical base pair must occur in the progeny molecules. However, many factors interfere with accurate replication of DNA. If an A should pair with C or G with T as a result of tautomerization, a point mutation (a change in one base pair) will result. Occasionally, a segment of DNA will be replicated more than once (duplication) or a segment may fail to be replicated (deletion). These and other aberrations in DNA replication do occur, but the mechanism of replication has evolved to minimize such mistakes.

Semiconservative Replication:
        The information in each strand of the double helix acts as the template for the construction of a new double-helical DNA molecule; this is called semiconservative replication since one old strand of DNA is paired with one new strand to produce the daughter DNA molecule. All DNA molecules in all organisms replicate semiconservatively. This basic fact was first proved by Matthew Meselson and Frank Stahl who labeled DNA in E. coli by growing cells for many generations in medium with a heavy isotope of nitrogen (15N). Cells were then shifted to medium with 14N and continued to grow exponentially. After one generation of growth in 14N medium, the DNA was isolated from a sample of cells and analyzed by cesium chloride density centrifugation. All DNA was of intermediate (hybrid) density; one strand contained JSN and the newly synthesized strand contained 14N. After two generations of growth in 14N medium, 50% of the DNA contained only 14N (light DNA) and 50% is hybrid. This key experiment confirmed a prediction of the Watson-Crick model for DNA. Subsequent experiments also explained that human DNA undergoes semiconservative replication.

Origin and Direction of DNA Replication:

       Another unifying principle of DNA replication is that each molecule of DNA has one or more specific origins of replication where DNA synthesis begins. In bacteria that carry their genetic information in a circular DNA molecule, only one origin of replication exists. Plasmid DNA also contains a unique origin of replication. Origins of replication can be moved from one location in DNA to another, or f rom one DNA molecule to another just as genes can be moved by genetic engineering techniques. In both bacterial and plasmid DNA, replication is initiated at a unique origin and proceeds in both directions along the DNA molecule to a termination site that is located approximately 180 degrees from the origin. Thus, replication of DNA is, in most cases, bidirectional replication. 

       The ability of DNA to replicate from many origins and in both directions means that cells can replicate all of their DNA in a fairly short period of time. The shortest generation time for the bacterium E. coli is approximately 30 minutes in rich medium in the laboratory whereas the shortest division time for a human cell is approximately 24 hours. Unwinding a double helix during replication presents a serious mechanical problem. Either the two daughter DNA molecules at the replication fork (the Y-shaped fork) must rotate around one another, or the unreplicated segment of DNA must rotate. This necessity for DNA rotation during replication creates topological problems for covalently closed circular DNA in bacteria and for the enormously long condensed and folded DNA in human chromosomes. The unwinding of strands of the DNA double helix is accomplished by a special group of enzymes called helicases; positive and negative coiling of DNA to maintain the topology necessary for replication is the function of another group of enzymes called topoisomerases.

Replicons:

        Much of the basic understanding of DNA replication was gleaned from studying the replication of plasmids: small, circular, extrachromosomal DNA elements in bacteria. Some plasmids, such as the F (fertility) factor in E. coli, have an origin of replication but replicate only unidirectionally from that single origin by a rolling-circle mechanism of replication. If an F factor integrates into the bacterial chromosome, it can replicate its own DNA and bacterial DNA as well during conjugation, a process in which bacteria physically join and transfer DNA from the donor bacterium to the recipient bacterium. In this situation, bacteria replicate DNA simultaneously in a bidirectional manner from their own origin of replication (oric) and unidirectionally from the origin of the integrated F factor (oriF). Such replication clearly raises topological problems during DNA replication. Studies of bacterial and plasmid DNA replication led to the concept of a replicon, which is:
1. A site on DNA consisting of a sequence of nucleotides that defines an origin of replication, and
2. Structural genes coding for proteins that recognize and bind to the origin to initiate DNA replication.
These genes and the segment of DNA that is replicated from the origin are collectively defined as a replicon. Thus, a bacterial chromosome or a plasmid is a single replicon, whereas a single human chromosome may contain thousands of replicons.
Discontinuous DNA Replication:
       DNA cannot replicate by copying both strands continuously because DNA polymerases, the enzymes that synthesize new DNA, can only add nucleotides to a 3’- OH group. Because of the antiparallel nature of the DNA strands, synthesis of one new strand of DNA can be continuous but synthesis along the other strand must occur by discontinuous replication. At each replication fork one DNA strand has a free 3'-OH group; the other has a free 5'-PO2- group. Since DNA can only elongate in a 5' to 3' direction, synthesis of one strand (the leading strand) is continuous; synthesis of the opposite strand (the lagging strand) is discontinuous. 

       Short fragments of DNA are synthesized in the replication fork on the lagging strand in a 5'to 3' direction; these are called Okazaki fragments after the scientist (Reiji Okazaki) who first demonstrated their existence during replication. These Okazaki fragments are joined in the replication fork by the enzyme DNA ligase that can form a phosphodiester bond at a single-strand break in DNA. DNA ligase joins a 3'-OH at the end of one DNA fragment to the 5'-monophosphate of the adjacent fragment. However, if even one nucleotide is missing in the DNA strand, ligase cannot seal the sugar-phosphate backbone.

Enzymology of DNA Replication:

         The fundamental enzymology of DNA replication derives from both in vivo and in vitro studies with cells and extracts derived from E. coli. Many of the enzymes involved in DNA replication were identified by isolation of conditional lethal mutants of the bacterium, e.g., mutants that are unable to replicate DNA (and unable to grow) at high temperatures (42 degrees C) but that replicate and grow normally at low temperatures (30 degrees C). The synthesis of DNA is a complex process because of the need for faithful replication, enzyme specificity, and topological constraints. Approximately 20 different enzymes are utilized in bacteria to replicate DNA. In addition to polymerization reactions, DNA replication requires accurate initiation, termination, and proofreading to eliminate errors.

DNA Polymerases:
        Three DNA polymerases have been characterized in E. coli and are designated polymerase I, polymerase II, and polymerase III. Although present in very low concentration in the cell, polymerase III, also called replicase, is the polymerase that elongates both strands of the bacterial DNA in the replication fork. Polymerase I is primarily a DNA repair enzyme and is responsible for excision of the short RNA primer that is required to initiate DNA synthesis on both the leading and lagging strands of DNA during replication. It also can remove mismatched base pairs during replication and fill in gaps in single stranded DNA that is joined in a double helix. 

       The function of polymerase II is not clear but it probably also has repair functions. All DNA polymerases select the nucleotide that is to be added to the 3'-OH end of the growing chain and catalyze formation of the phosphodiester bond. The substrates for DNA polymerases are the four deoxynucleoside-5'-triphosphates (dATE dCTP, dGTP, and dTTP) and a single-stranded template DNA. The overall chemical reaction catalyzed by all DNA polymerases is

Poly(nucleotide)n-3’-OH+dNTPà Poly(nucleotide n+1)-3’OH+PPi

in which PPi represents pyrophosphate cleaved from the dNTE That is, a reaction occurs between a 3'-OH group at a terminus of a DNA strand and the phosphoryl group (the one linked to the sugar) of an incoming nucleoside triphosphate. Even though the hydrolysis of the nucleoside triphosphates has a large negative A G, the polymerization reaction as written still has a positive A G at concentrations present in a cell and in laboratory reactions (+0.5 kcal/mol = 2.1 kJ/mol). Thus, in the absence of any
other reaction DNA polymerases would catalyze depolymerization rather than polymerization. Indeed, if excess pyrophosphate and a polymerase are added to a solution containing a partially replicating DNA molecule, the polymerase acts like a nuclease. In order to drive the reaction to the right, pyrophosphate must be removed, and this is ac complished by a potent pyrophosphatase, a widely distributed enzyme that breaks down pyrophosphate to inorganic phosphate. Hydrolysis of pyrophosphate has a large negative free energy, so essentially all of the pyrophosphate is rapidly removed. No DNA polymerase can catalyze the reaction between two free nucleotides, even if one has a 3'-OH group and the other a 5'-triphosphate. 

       Polymerization can occur only if the nucleotide with the 3'-OH group is hydrogen-bonded to the template strand. Such a nucleotide is called a primer. The primer can either be a single nucleotide or the terminus of a hydrogen-bonded oligonucleotide. When an incoming nucleotide is joined to a primer it supplies another free 3'-OH group, so that the growing strand itself is a primer. Since polymerization occurs only at the 3'-OH end, strand growth is said to proceed in the 5' -->3' direction. All known polymerases (both DNA and RNA) are capable of chain growth only in the 5' -->3' direction. This unidirectional feature of polymerases complicates the simultaneous replication of both strands of DNA. 

        Polymerization is not confined to addition of a nucleotide to a growing strand in a replication fork. For example, pol I can also add nucleotides to the 3'-OH group at a single-strand (a nick) in a double helix. This activity results from the ability of pol I both to recognize a 3’-OH group anywhere in the helix and to displace the base-paired strand ahead of the available 3'-OH group. This reaction is called strand displacement. Not all DNA polymerases can carry out strand displacement. Pol I has several enzymatic activities other than its ability to polymerize; one of these is important in maintaining continuity of the daughter strand, and the other improves the fidelity of replication. These two functions are a 5' -->3' exonuclease activity and a 3'-->5' exonuclease activity.
Exonuclease Activities of Polymerase I:
         Occasionally DNA polymerase, in error, adds a nucleotide to the 3'-OH terminus that cannot hydrogen-bond to the corresponding base in the template strand. Such a nucleotide would clearly change the information content of the daughter DNA molecule, and mechanisms exist for rectifying such incorporation errors. Once having added an incorrect nucleotide and moved on to the next position, pol I cannot add another nucleotide because the enzyme requires a primer that is correctly hydrogen-bonded. Where such an impasse is encountered, a 3' -->5' exonuclease activity, which may be thought of simply as pol I running backward or in the 3' -->5' direction, is stimulated and the unpaired base is removed. After removal of this base, the exonuclease activity stops, polymerizing activity is restored, and chain growth resumes. This exonuclease activity is called the proofreading or editing function of polymerase I. 

           Pol I also has a potent 5' -->3' exonucleolytic activity. This activity is directed against a basepaired strand and consists of stepwise removal of nucleotides one by one from the 5'-P terminus. Furthermore, the nucleotide removed can be either of the deoxyribo or the ribo type. The 5' -->3' exonucleolytic activity also can be coupled to the polymerizing function. Recall that pol I can add nucleotides to a 3'-OH group at a nick and displace the downstream strand. Under certain conditions, the displacement reaction does not occur and instead the 5' --->3' exonuclease acts on the strand that would otherwise be displaced, removing one downstream nucleotide for each nucleotide added to the 3' side of the nick. Thus, the position of the nick moves along the strand; this reaction is called nick translation. 

           It is a key laboratory procedure for synthesizing labeled DNA that can be used as probes; simply by carrying out the reaction in the presence of radioactive or chemically labeled nucleotides, an unlabeled DNA molecule with nicks in both strands can be converted to a labeled molecule. DNA probes are used for various diagnostic and forensic purposes. The main function of the 5' -->3' exonuclease activity is to remove ribonucleotide primers that are used in DNA replication. Pol I also possess a 5' -->3' endonuclease activity. An endonucleolytic cut is made between two base pairs that follow a 5'-P-terminated segment of unpaired bases. This type of endonucleolytic activity is unimportant in normal replication but is important in excision repair.

Polymerase III:
         Polymerase I plays an important role in the replication process in E. coli, but it is not responsible for the entire polymerization of the replicating strands. The enzyme that performs this is a less abundant enzyme, polymerase III (pol III). A DNA polymerase II has also been extracted from E. coli, but it probably plays no role in DNA synthesis. Pol III catalyzes the same polymerization reaction as pol I but contain certain different features. It is a very complex enzyme and is associated with eight other proteins to obtain the pol III holoenzyme. 

         Pol III is similar to pol I in that it has a requirement for a template and a primer but its substrate specificity is much more limited. For a template pol III cannot act at a nick nor can it unwind a helix and carry out strand displacement. The latter deficiency means that an auxiliary system is required to unwind the helix ahead of a replication fork. Pol III, like pol I, possesses a 3' -->5' exonuclease activity, which performs the major editing function in DNA replication. Polymerase III also has a 3' exonuclease activity, but this activity does not appear to play a role in replication.

         Pol I and pol III holoenzyme are important for E. coli replication. The need for two polymerases appears to be characteristic of all cellular organisms but not all viruses, e.g., E. coli. Phage T4 synthesizes its own DNA polymerase, which is able to carry out all functions necessary for synthesizing phage DNA. In the general polymerization reaction, the activation energy for phosphodiester bond formation arises from cleaving of the triphosphate. Since DNA ligase can use a monophosphate, another source of energy is required. 

         This energy is obtained by hydrolyzing either ATP or NAD; the energy source depends on the organism from which the DNA ligase is obtained. Ligases have two major functions: the sealing of singlestrand breaks produced randomly in DNA molecules by nucleases and the joining of fragments during a particular stage of replication. DNA ligases are enzymes that are able to form a phosphodiester bond at a single-strand break in DNA, a reaction between a 3'-OH group and a 5'-monophosphate. 

         These groups must be termini of adjacent base-paired deoxynucleotides. Bacteria usually contain a single species of ligase. Mammalian cells possess two DNA ligases (I and II) present in very small amounts compared with bacteria. Both eukaryotic ligases are located in the nucleus. Ligase I is predominant in proliferating cells and presumably plays a role in DNA replication; ligase II predominates in resting cells.

Eukaryotic Polymerases:

         Five polymerizing enzymes have been isolated from many mammalian cells. Three-pol α, pol β, and pol γ function in replication. Pol α is the major polymerase of mammalian cells; it is found in the nuclei and is analogous to E. coli pol III. It is a multi subunit enzyme with a core (4-5 subunits) responsible for polymerization and a holoenzyme form possessing additional subunits and activities. It lacks the 3' -->5' exonuclease editing activity. An intriguing protein subunit in the holoenzyme enables it to bind AppppA (diadenosine tetraphosphate), a small molecule that stimulates replication of resting mammalian cells and is hypothesized to be a growth signal. The pol α holoenzyme of rat liver also possesses a DNA primase activity, a feature not found in prokaryotic enzymes. Pol β is a nuclear polymerase, probably analogous in function to E. coli pol I. Pol y is found in mitochondria and is responsible for replication of mitochondrial DNA. It functions in the same way as pol III but is a single polypeptide.

The Replication Fork:

         DNA replication requires not only an enzymatic mechanism for adding nucleotides to the growing chains but also a means of unwinding the parental double helix. These are distinct processes, and the unwinding of the helix is closely related to the initiation of synthesis of precursor fragments. The pol III holoenzyme cannot unwind the helix. In order for unwinding to occur, hydrogen bonds and hydrophobic interactions must be eliminated, which requires energy. Pol I utilizes the free energy of hydrolysis of the triphosphate for unwinding as it synthesizes a DNA strand in a way that other polymerases cannot; instead, helix unwinding is accomplished by enzymes called helieases. These enzymes hydrolyze ATP and utilize the free energy of hydrolysis for unwinding. 

          Unwinding of the helix by a helicase is not sufficient in itself for advance of the replication fork. Accessory proteins called single-stranded DNA-binding proteins (SSB proteins) are usually needed. As a helicase advances, it leaves in its wake two single-stranded regions: a longer one that is copied discontinuously and a shorter one just ahead of the leading strand. In order to prevent the single-stranded regions from reannealing or from forming intrastrand hydrogen bonds, the single-stranded DNA is protected with SSB proteins. SSB proteins bind tightly to both single-stranded DNA and to one another and hence are able to cover extended regions. 

            As the polymerase advances, it must displace the SSB proteins so that base pairing of the added nucleotide can occur. Some phage replication systems utilize a single protein that functions as both a helicase and an SSB protein; the gene-32 protein of phage T4 is the prototype. It binds very tightly to single-stranded DNA and exceedingly tightly to itself, and its binding energy is great enough to unwind the helix. As a replication fork moves along a circular helix, rotation of the daughter molecules around one another causes the individual polynucleotide strands of the unreplicated portion of the molecule to become wound more tightly, i.e., overwound. This may be difficult to visualize but can be seen by taking two interwound circular strings and pulling them apart at any point. Thus, advance of the replication forks causes positive supercoiling of the unreplicated portion. 

            This supercoiling obviously cannot increase indefinitely because soon the unreplicated portion becomes coiled so tightly that further advance of the replication fork is impossible. 

This additional coiling can be removed by a topoisomerase that can introduce negative superhelicity. In E. coli, the enzyme DNA gyrase produces negative superhelicity in nonsupercoiled covalent circles and is also responsible for removing the positive superhelicity generated during replication. The evidence for this comes from in vivo experiments using drugs that inhibit DNA gyrase; addition of any of these drugs to a growing bacterial culture or raising the temperature of cells with a temperature-sensitive gyrase protein inhibits DNA synthesis. Furthermore, in vitro replication of circular DNA can proceed only if DNA gyrase or a similar topoisomerase is present in the reaction mixture.

Chromosome Replication:

           DNA in mammalian cells is organized in complex structures called chromosomes (prokaryotes do not have a nucleus, do not divide by mitosis, and do not, strictly speaking, have chromosomes). The DNA in the chromosomes of human and other eukaryotic cells is intimately associated with two classes of proteins called histones and nonhistones. Collectively, DNA, histones, and nonhistones constitute chromatin, from which the name chromosome is derived. The DNA in a chromosome is an extremely long, linear molecule that must be condensed and organized to fit into the chromosomes in the nucleus. The DNA in the 46 human chromosomes would be about 1 m long if fully extended. Histones are responsible for the structural organization of DNA in chromosomes; the nonhistone proteins reg ulate the functions of DNA including replication and gene expression. 

          The positive charge of histones, due to the presence of numerous lysine and arginine residues, is a major feature of the molecules, enabling them to bind to the negatively charged phosphate groups in DNA. The electrostatic attraction is an important stabilizing force in chromatin. If chromosomes are placed in solutions of high salt that break down electrostatic interactions, chromatin dissociates into free histones and DNA. Chromatin also can be reconstituted by mixing purified histones and DNA in concentrated salt solutions and gradually removing the salt by dialysis. Histones share a similar primary structure among eukaryotic species. However, they undergo various posttranslational modifications such as phosphorylation, acetylation, methylation, and ADP ribosylation.The chemical modifications of histones can alter their net charge, shape, and other properties affecting DNA binding. Pairs of four different histones (H2A, H2B, H3, and H4) combine to form an eight-protein bead around which DNA is wound; this bead-like structure is called a nucleosome. 

            A nucleosome has a diameter of 10 nm and contains approximately 200 base pairs. Each nucleosome is linked to an adjacent one by a short segment of DNA (linker) and another histone (H1). The DNA in nucleosomes is further condensed by the formation of thicker structures called chromatin fibers, and ultimately DNA must be condensed to fit into the metaphase chromosome that is observed at mitosis. Despite the dense packing of DNA in chromosomes, it must be accessible to regulatory proteins during replication and gene expression. At a higher level of organization, chromosomes are divided into regions called euchromatin and heterochromatin. Transcription of genes seems to be confined mainly to euchromatic regions while DNA in heterochromatic regions is genetically inactive. When DNA is replicated during the S phase of the cell cycle, the histone and nonhistone proteins also are duplicated and combine with the daughter DNA molecules.

Replication of DNA in Chromosomes:

           The rate of movement of a replication fork in E. coli is about 105 nucleotides per minute; in eukaryotes the DNA polymerases move only about one-tenth as fast. To replicate the entire DNA in a human cell in a few hours means that replication must be initiated at thousands of origins of replication in each chromosome and move bidirectionally. To accomplish this, mammalian cells have thousands of times more DNA polymerase available than is found in bacteria. As replication proceeds through each nucleosome, the histones must dissociate to allow the replication fork to proceed; after replication, the nucleosome re-forms. The separation of parental and daughter DNA also requires the synthesis of new nucleosomes. Recent studies suggest that DNA is replicated in "replication factories," i.e., fixed sites within the nucleus consisting of the numerous proteins needed for replication. The DNA is replicated by being drawn through the replication factories rather than having the replication proteins move along the DNA.
Replication at the Ends of Chromosomes:
           Since DNA in chromosomes is a linear molecule, problems arise when replication comes to the ends of the DNA. Synthesis of the lagging strand at each end of the DNA requires a primer so that replication can proceed in a 5' to 3' direction. This becomes impossible at the ends of the DNA and 50-100 bp is lost each time a chromosome replicates. Thus, at each mitosis of a somatic cell, the DNA in chromosomes becomes shorter and shorter. Ultimately, after a limited number of divisions, a cell enters a nondividing state, called replicative senescence, which may play an important role in biological aging. To prevent the loss of essential genetic information during replication, the ends of DNA in chromosomes contain special structures called telomeres that are synthesized by a specific enzyme called telomerase. Intact telomerase consists of an RNA primer and associated proteins so telomerase is actually a reverse transcriptase. 
           The activity of telomerase in replenishing telomeres is regulated by a number of telomere-specific DNA-binding proteins, TRF1 and TRF2. TRF1 regulates the length of telomeres and TRF2 protects the ends. Overexpression of TRFI results in progressive shortening of telomeres; underexpression results in lengthening. Another telomere regulatory protein is tankrase (TRFl-interacting ankyrin-related ADPribose polymerase), which alters the activity of TRF1. Telomeres in human chromosomes consist of tandem repeats of the sequence TTAGGG. In most adult somatic cells, telomerase activity is very low or absent. Even in hematopoietic stem cells that do have residual telomerase activity, telomere shortening is observed at the level of granulocyte and mononuclear cell fractions. 
             Accelerated telomere shortening has been observed in cells from patients with aplastic anemia, suggesting that abnormal telomere shortening is associated with disease and aging. A characteristic of malignant tumor cells is that they can replicate indefinitely. The immortality of tumor cells appears to result, at least in part, from enhanced levels of telomerase that allow them to repair and elongate telomeres at the ends of DNA. This hypothesis is supported by the observation that ectopic expression of the catalytic subunit of telomerase (a product of the hTERT gene) enabled human retinal pigmented epithelial cells and fibroblasts to avoid senescence and to maintain their differentiated
state when grown in vitro. Current research is focused on drugs that can promote or inhibit the action of telomerase or telomere-associated regulatory proteins. It is hoped that increasing telomerase activity in cells approaching senescence will retard aging or that decreasing telomerase activity in tumor cells will result in arrested tumor growth.