What is DNA replication?

DNA Structure and Function

The importance of chromosomes in heredity has been known since early in the twentieth century. Chromosomes consist of both DNA and protein, and in the early twentieth century there was considerable controversy concerning which component was the hereditary molecule. Early evidence favored the proteins. In 1944, however, a series of classic experiments by Oswald Avery, Maclyn McCarty, and Colin MacLeod lent strong support to the proponents favoring DNA as the genetic material. They showed that a genetic transforming agent of bacteria was DNA and not protein. In experiments reported in 1952, Alfred Hershey and Martha Chase provided evidence that DNA was the genetic material of bacteriophages (viruses that infect bacteria). Combined with additional circumstantial evidence from many sources, DNA became favored as the hereditary molecule, and a heated race began to determine its molecular structure.

In 1953, James Watson and Francis Crick published a model for the atomic structure of DNA. Their model was based on known chemical properties of DNA and x-ray diffraction data obtained from Rosalind Franklin
and Maurice Wilkins. The structure itself made it clear that DNA was indeed the molecule of heredity and provided evidence for how it might be copied. The molecule resembles a ladder. The “rails” are composed of repeating units of sugar and phosphate, forming a backbone for the molecule. Each “rung” consists of a pair of nitrogenous bases, one attached to each of the two rails and held together in the middle through weak bonds called hydrogen bonds. Since there are thousands to hundreds of millions of units on a DNA molecule, the hydrogen bonds between each pair of bases add up to a strong force that holds the two strands together. DNA, then, consists of two strands, each consisting of a repeating sugar-phosphate backbone and nitrogenous bases with the two strands held together by base-pair interactions. The two strands are oriented in opposite directions. The ends of a linear DNA molecule can be distinguished by which part of the backbone sugar is exposed and are referred to as the 5′ (five prime) end and the 3′ end, named for a particular carbon atom on the ribose sugar. If one DNA strand is oriented 5′ to 3′, its complementary partner is oriented 3′ to 5′. This organization has important implications for the mechanism of DNA replication.

There are four different bases:
adenine (A), guanine (G), cytosine (C), and thymine (T). They can be arranged in any order on a DNA strand, allowing the enormous diversity necessary to encode the blueprint of every organism. A key feature of the double-stranded DNA molecule is that bases have strict pairing restrictions: A can only pair with T; G can only pair with C. Thus if a particular base is known on one strand, the corresponding base is automatically known on the other. Each strand can serve as a template, or mold, dictating the precise sequence of bases on the other. This feature is fundamental to the process of DNA replication.

The genome (the complete DNA content of an organism) stores all the genetic information that determines the features of that organism. The features are expressed when the DNA is transcribed to a messenger molecule, mRNA, which is used to construct a protein. The proteins encoded by the organism’s genes in its DNA carry out all of the activities of the cell.

The Cell Cycle

In eukaryotic organisms (most organisms other than bacteria), cells progress through a series of four stages between cell divisions. The stages begin with a period of growth (G1 phase), followed by replication of the DNA (S phase). A second period of growth (G2 phase) is followed by division of the cell (M phase). Each of the two cells resulting from the cell division goes through its own cell cycle or may enter a dormant stage (G0 phase). The passage from one stage to the next is tightly regulated and directed by internal and external signals to the cell.

The transition from G1 into S phase marks the beginning of DNA replication. In order to enter S phase, the cell must pass through a checkpoint or restriction point in which the cell determines the quality of its DNA: If there is any damage to the DNA, entry into S phase will be delayed. This prevents the potentially lethal process of beginning replication of a DNA molecule that has damage that would prevent completion of replication. If conditions are determined to be acceptable, a “molecular switch” is thrown, triggering the initiation of DNA replication. What is the nature of this molecular switch? There are many different proteins that participate in the process of DNA replication, and they can have their activity turned off and on by other proteins. Addition or removal of a chemical group called a phosphate is a common mechanism of chemical switching. This reaction is catalyzed by a class of enzymes called kinases. Certain key proteins are phosphorylated at the boundary of the G1 and S phases of
the cell cycle by kinases, switching on DNA replication.

Origins and Initiation

If the human genome were replicated from one end to the other, it would take several years to complete the process. The DNA molecule is simply too large to be copied end to end. Instead, replication is initiated at many different sites called origins of replication, and DNA synthesis proceeds from each site in both directions until regions of copied DNA merge. The region of DNA copied from a particular origin is called a replicon. Using hundreds to tens of thousands of initiation sites and replicons, the genome can be copied in a matter of hours. The structure of replication origins has been difficult to identify in all but a few organisms, most notably yeast. Origins consist of several hundred base pairs of DNA comprising sequences that attract and bind a set of proteins called the origin recognition complex (ORC). The exact mechanism by which the origin is activated is still under investigation, but a favored model is supported by all of the available evidence.

The ORC proteins are believed to be bound to the origin DNA throughout the cell cycle but become activated at the G1/S boundary through the action of kinases. Kinases add phosphate groups to one or more of the six ORC proteins, activating them to initiate DNA replication. Different replicons are initiated at different times throughout S phase. It is unclear how the proposed regulatory system distinguishes between replicons that have been replicated in a particular S phase and those that have not, since each must be used once and only once during each cell division cycle.

A number of different enzymatic activities are required for the initiation process. The two strands of DNA must be unwound or separated, exposing each of the parent strands so they can be used as templates for the synthesis of new, complementary strands. This unwinding is mediated by an enzyme called a helicase. Once unwound, the single strands are stabilized by the binding of proteins called single-strand binding proteins (SSBs). The resulting structure resembles a “bubble” or “eye” in the DNA strand. This structure is recognized by the DNA replication machinery that is recruited to the site, and DNA replication begins. As replication proceeds, the DNA continues to unwind through the action of helicase. The site at which unwinding and DNA synthesis are occurring is at either end of the expanding eye or bubble, called a replication fork.

DNA Synthesis

The DNA synthesis machinery is not able to synthesize a strand of DNA from scratch; rather, a short stretch of RNA is used to begin the new strands. The synthesis of the RNA is catalyzed by an enzyme called primase. This short piece of RNA, or primer, is extended using DNA nucleotides by the enzymes of DNA synthesis, called DNA polymerases. The RNA primer is later removed and replaced by DNA. Nucleotide monomers align with the exposed template DNA strand one at a time and are joined by the DNA polymerase. The joining of nucleotides into a growing DNA chain requires energy. This energy is supplied by the nucleotide monomers themselves. A high-energy phosphate bond in the nucleotide is split, and the breakage of this high-energy bond provides the energy to drive the polymerase reaction.

The two strands of DNA are not synthesized in the same way. The two strands are oriented opposite one another, but DNA synthesis only occurs in one direction: 5′ to 3′. Therefore, one strand, called the leading strand, is synthesized continuously in the same direction that the replication fork is moving, while the lagging strand is synthesized away from the direction of fork movement. Since the lagging-strand DNA synthesis and fork movement are in opposite directions, this strand of DNA must be made in short pieces that are later joined. Lagging-strand synthesis is therefore said to be discontinuous. These short intermediates are called Okazaki fragments, named for their discoverer, Reiji Okazaki. Overall, DNA replication is said to be semidiscontinuous.

The DNA synthesis machine operating at the replication fork is a complex assembly of proteins. Many different activities are necessary to carry out the process of DNA replication efficiently. Several proteins are necessary to recognize the unwound origin and assemble the rest of the complex. Primase must function to begin both new strands and is then required periodically throughout synthesis of the lagging strand. A doughnut-shaped clamp called PCNA functions as a “processivity factor” to keep the entire complex attached to the DNA until the job is completed. Helicase is continuously required to unwind the template DNA and move the fork along the parent molecule. As the DNA is unwound, strain is created on the DNA ahead of the replication fork. This strain is alleviated through the action of topoisomerase enzymes. Single-strand binding proteins are needed to stabilize the regions of unwound DNA that exist before the DNA is actually copied. Finally, an enzyme called ligase is necessary to join the regions replicated from different origins and to attach all of the Okazaki
fragments of the lagging strand. All of these proteins are part of a well-orchestrated, efficient machine ideally suited to its task of copying the genetic material.

DNA polymerases are not perfect. At a relatively low frequency, they can add an incorrect nucleotide to a growing chain, one that does not match the template strand as dictated by the base-pairing rules. However, because the DNA molecules are so extremely large, novel mechanisms for proofreading have evolved to ensure that the genetic material is copied accurately. DNA polymerases can detect the misincorporation of a nucleotide and use an additional enzymatic activity to correct the mistake. Specifically, the polymerase can “back up” and cut out the last nucleotide added, then try again. With this and other mechanisms to correct errors, the observed error rate for DNA synthesis is a remarkable one error in every billion nucleotides added.

Impact and Applications

DNA replication is a fundamental cellular process: Proper cell growth cannot occur without it. It must be carefully regulated and tightly controlled. Despite its basic importance, the details of the mechanisms that regulate DNA replication are poorly understood. Even with all of the checks and balances that have evolved to ensure a properly replicated genome, occasional mistakes do occur. Attempting to replicate a genome damaged by chemical or other means may simply lead to death of a single cell. Far more ominous are genetic errors that lead to loss of regulating mechanisms. Without regulation, cell growth and division can proceed without normal limits, resulting in cancer. Much of the focus for the study of cell growth and regulation is to set a foundation for the understanding of how cancer cells develop. This knowledge may lead to new techniques for selective inhibition or destruction of cancer cells.

Manipulation of DNA replication and cell cycle control are the newest tools for progress in genetic engineering. In early 1997, the first successful cloning of an adult mammal, Dolly the sheep, raised important new issues about the biology and ethics of manipulating mammalian genomes. The technology now exists to clone human beings, although such experiments are not likely to be carried out. More relevant is the potential impact on agriculture. It is now possible to select for animals that have the most desirable traits, such as lower fat content or disease resistance, and create herds of genetically identical animals. Of direct relevance to humans is the potential impact on the understanding of fertility and possible new treatments for infertility.

A new class of genetic diseases was discovered in the 1980s called triplet repeat diseases. Regions of DNA consist of copies of three nucleotides (such as CGG) that are repeated up to fifty times. Through unknown mechanisms related to DNA replication, the number of repeats may increase from generation to generation, at some point reaching a threshold level at which disease symptoms appear. Diseases found to conform to this pattern include fragile X syndrome, Huntington disease (Huntington chorea), and Duchenne muscular dystrophy.

The process of aging is closely related to DNA replication. Unlike bacteria, eukaryotic organisms have linear chromosomes. This poses problems for the cell, both in maintaining intact chromosomes (ends are unstable) and in replicating the DNA. The replication machinery cannot copy the extreme ends of a linear DNA molecule, so organisms have evolved alternate mechanisms. The ends of linear chromosomes consist of telomeres (short, repeated DNA sequences that are bound and stabilized by specific proteins), which are replicated by a separate mechanism using an enzyme called telomerase. Telomerase is inactivated in mature cells, and there may be a slow, progressive loss of the telomeres that ultimately leads to the loss of important genes, resulting in symptoms of aging. Cancer cells appear to have reactivated their telomerase, so potential anticancer therapies are being developed based on this information.

Key Terms



replication

:

the process by which one DNA molecule is converted to two DNA molecules identical to the first




transcription

:

the process of forming an RNA according to instructions contained in DNA




translation

:

the process of forming proteins according to instructions contained in an RNA molecule





x-ray diffraction


:

a method for determining the structure of molecules which infers structure by the way crystals of molecules scatter X rays as they pass through





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