Replication

Overview

The DNAs that make up the genomes of bacteria and eukaryotic cells are double-stranded molecules in which each strand is composed of subunits called nucleotides. DNA nucleotides have a direction, in the same way that an arrow has a head and a tail. In DNA strands, the head is the 3′ ("three prime") end of the strand, and the tail is the 5′ ("five prime") end. As a result, each strand also has a direction, whose ends are referred to as the 3′ and 5′ ends. The two strands of DNA run in opposite directions, and are wound around each other in a double helix, with the strands held together by hydrogen bonds between paired bases of the nucleotides (A pairs with T, and G pairs with C).

During the process of DNA replication, the strands are unwound by an enzyme called DNA helicase, and a new strand of DNA is synthesized on each of the old (template) strands by an enzyme called DNA polymerase, which joins incoming nucleotides together in a sequence that is determined by the sequence of nucleotides present in the template strand. DNA replication is said to be semiconservative because each of the two identical daughter molecules contains one of the two parental template strands paired with a new strand. Prokaryotic replication can take as little as twenty minutes, while replication in eukaryotes takes considerably longer, approximately eight hours in mammals.

Initiation of DNA Replication

DNA replication begins (initiates) at special sites called origins of DNA replication. Eukaryotic DNAs each contain multiple replication origins, spaced at intervals of approximately 100,000 base pairs (100 kilobase pairs, or 100 kb) along the length of the DNA. There are 6 billion base pairs in the human genome, located on forty-six chromosomes, and so each chromosome will have many origins of replication. Prokaryotic chromosomes typically have a single replication origin.

Replication origins are composed of special sequences of DNA that are recognized by replication initiator proteins, which bind to the origin sequences and then help to assemble other proteins required for DNA replication at these sites. The eukaryotic replication initiator protein is a complex containing six different subunits called the origin recognition complex (ORC). The bacterial replication initiator protein is called the dna A protein. The timing of DNA replication is regulated by controlling the assembly of complexes at replication origins.

The distinct steps in the initiation of replication are understood better in bacteria than in eukaryotes, but several key steps are common to both. The first step is a change in the conformation of the initiator protein, which causes limited "melting" (that is, the separation of the two strands) of the double-stranded DNA next to the initiator binding site, thus exposing single-stranded regions of the template (Figure 1). Two more proteins, DNA helicase and DNA primase, then join the complex. Replication initiation is triggered by the activation of the helicase and primase, and the subsequent recruitment of DNA polymerase. In prokaryotes, the particular form of the enzyme is called DNA polymerase III. Other proteins are also recruited, each of whose functions are discussed below.

The Replication Fork

The separation of the two template strands and the synthesis of new daughter DNA molecules creates a moving "replication fork" (Figure 2), in which, double-stranded DNA is continually unwound and copied. The unwinding of DNA poses special problems, which can be visualized by imagining pulling apart two pieces of string that are tightly wound around each other. The pulling apart requires energy; the strands tend to rewind if not held apart; and the region ahead of the separated strands becomes even more tightly twisted.

Proteins at the replication fork address each of these problems. DNA polymerases are not able to unwind double-stranded DNA, which requires energy to break the hydrogen bonds between the bases that hold the strands together. This task is accomplished by the enzyme DNA helicase, which uses the energy in ATP to unwind the template DNA at the replication fork. The single strands are then bound by a single-strand binding protein (called SSB in bacteria and RPA in eukaryotes), which prevents the strands from reassociating to form double-stranded DNA. Unwinding the DNA at the replication fork causes the DNA ahead of the fork to rotate and become twisted on itself. To prevent this from happening, an enzyme called DNA gyrase (in bacteria) or topoisomerase (in eukaryotes) moves ahead of the replication fork, breaking, swiveling, and rejoining the double helix to relieve the strain.

Leading Strands and Lagging Strands

The coordinated synthesis of the two daughter strands posed an important problem in DNA replication. The two parental strands of DNA run in opposite directions, one from the 5′ to the 3′ end, and the other from the 3′ to the 5′ end. However, all known DNA polymerases catalyze DNA synthesis in only one direction, from the 5′ to the 3′ end, adding nucleotides only to the 3′ end of the growing chain. The daughter strands, if they were both synthesized continuously, would have to be synthesized in opposite directions, but this is known not to occur. How, then, can the other strand be synthesized?

The resolution of the problem was provided by the demonstration that only one of the two daughter strands, called the leading strand, is synthesized continuously in the overall direction of fork movement, from the 5′ to the 3′ end (see Figure 3). The second daughter strand, called the lagging strand, is made discontinuously in small segments, called Okazaki fragments in honor of their discoverer. Each Okazaki fragment is made in the 5′ to 3′ direction, by a DNA polymerase whose direction of synthesis is backwards compared to the overall direction of fork movement. These fragments are then joined together by an enzyme called DNA ligase.

The Need for Primers

Another property of DNA polymerase poses a second problem in understanding replication. DNA polymerases are unable to initiate synthesis of a new DNA strand from scratch; they can only add nucleotides to the 3′ end of an existing strand, which can be either DNA or RNA. Thus, the synthesis of each strand must be started (primed) by some other enzyme.

The priming problem is solved by a specialized RNA polymerase, called DNA primase, which synthesizes a short (3 to 10 nucleotides) RNA primer strand that DNA polymerase extends. On the leading strand, only one small primer is required at the very beginning. On the lagging strand, however, each Okazaki fragment requires a separate primer.

Before Okazaki fragments can be linked together to form a continuous lagging strand, the RNA primers must be removed and replaced with DNA. In bacteria, this processing is accomplished by the combined action of RNase H and DNA polymerase I. RNase H is a ribonuclease that degrades RNA molecules in RNA/DNA double helices. In addition to its polymerase activity, DNA polymerase I is a 5′-to-3′ nuclease, so it too can degrade RNA primers. After the RNA primer is removed and the gap is filled in with the correct DNA, DNA ligase seals the nick between the two Okazaki fragments, making a continuous lagging strand.

DNA Polymerase

The two molecules of DNA polymerase used for the synthesis of both leading and lagging strands in bacteria are both DNA polymerase III. They are actually tethered together at the fork by one of the subunits of the protein, keeping their progress tightly coordinated. Many of the other players involved are also linked, so that the entire complex functions as a large molecular replicating machine.

DNA polymerase III has several special properties that make it suitable for its job. Replication of the leading strand of a bacterial chromosome requires the synthesis of a DNA strand several million bases in length. To prevent the DNA polymerase from "falling off" the template strand during this process, the polymerase has a ring-shaped clamp that encircles and slides along the DNA strand that is being replicated, holding the polymerase in place. This sliding clamp has to be opened like a bracelet in order to be loaded onto the DNA, and the polymerase also contains a special clamp loader that does this job.

A second important property of DNA polymerase III is that it is highly accurate. Any mistakes made in incorporating individual nucleotides cause mutations, which are changes in the DNA sequence. These mutations can be harmful to the organism. The accuracy of the DNA polymerase results both from its ability to select the correct nucleotide to incorporate, and from its ability to "proofread" its work.

Appropriate nucleotide selection depends on base-pairing of the incoming nucleotide with the template strand. At this step, the polymerase makes about one mistake per 1,000 to 10,000 incorporations. Following incorporation, the DNA polymerase has a way of checking to see that the nucleotide pairs with the template strand appropriately (that is, A only pairs with T, C only pairs with G). In the event that it does not, the DNA polymerase has a second enzymatic activity, called a proofreading exonuclease, or a 3′-to-5′ exonuclease, that allows it to back up and remove the incorrectly incorporated nucleotide. This ability to proofread reduces the overall error rate to about one error in a million nucleotides incorporated. Other mechanisms detect and remove mismatched base pairs that remain after proofreading and reduce the overall error rate to about one error in a billion.

Features of Replication in Eukaryotic Cells

The steps in DNA replication in eukaryotic cells are very much the same as the steps in bacterial replication discussed above. The differences in bacterial and eukaryotic replication relate to the details of the proteins that function in each step. Although amino acid sequences of eukaryotic and prokaryotic replication proteins have diverged through evolution, their structures and functions are highly conserved. However, the eukaryotic systems are often somewhat more complicated.

For example, bacteria require only a single DNA polymerase, using DNA polymerase III for both leading and lagging strand synthesis, and are able to survive without DNA polymerase I. In contrast, eukaryotes require at least four DNA polymerases, DNA polymerases α, δ, ε, and σ. DNA polymerases δ and ε both interact with the sliding clamp, and some evidence suggests that one of these polymerases is used for the leading strand and the other for the lagging strand. One required function of DNA polymerase σ is the synthesis of the RNA primers for DNA synthesis. The precise role of DNA polymerase is not yet known. A second example is removal of the RNA primers on Okazaki fragments. In eukaryotes, primer removal is carried out by RNase H and two other proteins, Fen1 and Dna2, which replace the 5′-to-3′ exonuclease provided by the bacterial DNA polymerase I in bacteria.

Replication continues until two approaching forks meet. The tips of linear eukaryotic chromosomes, called telomeres, require special replication events. Bacterial chromosomes, which contain circular DNA molecules, do not require these special events.

Regulating Replication

DNA replication must be tightly coordinated with cell division, so that extra copies of chromosomes are not created and each daughter cell receives exactly the right number of each chromosome. DNA replication is regulated by

controlling the assembly of complexes at replication origins. In bacteria, the accumulation of the initiator protein, dnaA, seems to be an important factor in determining when replication begins.

In eukaryotes, DNA replication and cell division are separated by two "gap" cell cycle phases (G₁ and G₂), during which neither DNA replication nor nuclear division occurs. DNA replication occurs during the S (or synthesis) phase, but ORC is thought to bind replication origins throughout the cell cycle. During the G₁ phase of the cell cycle, ORC helps to assemble other replication initiation factors at replication origins to make so-called pre-replicative-complexes (pre-RCs) that are competent to initiate replication during S phase. These other initiation factors include a protein called Cdc6 and a family of six related MCM ("mini-chromosome maintenance") proteins. The functions of these proteins are not yet known; however, the MCM proteins are currently the best candidate for the eukaryotic replicative helicase, and Cdc6 is necessary for MCM proteins to bind DNA. DNA polymerase also assembles on origins during this time.

Replication initiation is actually triggered at the beginning of S phase by the phosphorylation (addition of a phosphate group to) of one or more proteins in the pre-RC. The enzymes that phosphorylate proteins in the pre-RC are called protein kinases. Once they become active, they not only trigger replication initiation, but they also prevent the assembly of new pre-RCs. Therefore, replication cannot begin again until cells have completed cell division and entered G₁ phase again.

SEE ALSO CELL CYCLE; CHROMOSOME, EUKARYOTIC; CHROMOSOME, PROKARYOTIC; DNA; DNA POLYMERASES; DNA REPAIR; MUTATION; NUCLEASES; NUCLEOTIDE; NUCLEUS; TELOMERE.

Carol S. Newlon

Bibliography

Baker, T. A., and S. P. Bell. "Polymerases and the Replisome: Machines within Machines." Cell 92 (1998): 295-305.

Cooper, Geoffrey M. The Cell: A Molecular Approach. Washington, DC: ASM Press, 1997.

Herendeen, D. R., and T. J. Kelly. "DNA Polymerase III: Running Rings Around the Fork." Cell 84 (1996): 5-8.

Lodish, Harvey, et al. Molecular Cell Biology, 4th ed. New York: W. H. Freeman, 2000.

Stillman, B. "Cell Cycle Control of DNA Replication." Science 274 (1996): 1659-1664.

Internet Resource

Davey, M., and M. O'Donnell. "DNA Replication." Genome Knowledge Base Website. <http://gkb.cshl.org/db/index>.