DNA Replication Overview

This module looks at the way DNA reproduces itself, which is one of the necessary properties for its role as hereditary material.

Objectives
1. Understand the semiconservative nature of DNA replication. Realize that the process begins at unique origins of replication, and proceeds bidirectionally. 2. Know that DNA synthesis is catalyzed by a family of enzymes called DNA polymerases. Understand that DNA polymerase has a requirement for a template on which to synthesize the new DNA strand, and for a primer from which to extend the DNA strand. 3. Understand the various functions of the RNA polymerases, such as exonuclease and polymerase activities, and their function in the replication process. 4. Know the other components of the replication apparatus, and their basic functions. Understand the principles of leading strand synthesis and lagging strand synthesis. 5. Realize that eukaryotes require the activity of telomerase to complete the synthesis of their linear chromosomes. The Semiconservative Nature of DNA Replication One property of the genetic material necessary for its function is the ability to replicate (reproduce) itself. After it was established that DNA is the genetic material, attention turned toward how DNA was replicating in living organisms. The Watson-Crick model of DNA structure (as outlined in the module on nucleic acids) suggested a possible mechanism for replication of DNA molecules. The nature of base pairing meant that if the two strands of a DNA molecule were separated, they could each serve as a template for the creation of a complementary strand by bringing in individual nucleotides to base pair with their complementary base on the template, and joining the new nucleotides together. Thus, each DNA molecule after replication would consist of one of the original strands plus one newly synthesized strand. This model of DNA replication is called semiconservative. Semiconservative was not the only model of DNA replication, however. Other proposed models included conservative replication and dispersive replication. Conservative replication proposed that after replication, one DNA molecule consists entirely of newly synthesized DNA whereas the other molecule is entirely original DNA. Dispersive replication suggested that each DNA molecule after replication might consist of segments of new and old DNA interspersed. It would be difficult to devise a mechanism by which

this latter outcome might occur, but until evidence to the contrary was produced, it had to be considered. The three possible models of DNA replication are depicted below.

To distinguish between these possibilities, Meselson and Stahl did the following experiment: First, they grew bacteria for many generations in a growth medium containing 15N. This is a heavy isotope of nitrogen (in contrast to the normal isotope, 14 N), which over many generations would be incorporated into all nitrogen-containing molecules of the cells, including DNA. DNA isolated from these cells could be distinguished from normal DNA because it would have a higher density. The bacteria grown in heavy nitrogen were then transferred to growth medium containing 14 N for one round of replication. This lighter isotope would incorporate into any newly synthesized DNA. If semiconservative replication occurred, then each DNA molecule after replication would contain heavy nitrogen and light nitrogen, and would therefore have a density intermediate between the two. Conservative replication would produce one DNA molecule containing heavy nitrogen and one molecule containing light nitrogen, so there would be two different densities. Dispersive replication would produce a single intermediate density, just like semiconservative. The observed density of the DNA after one round of replication was intermediate. Replication was therefore either semiconservative or dispersive. These possibilities could be distinguished after a second round of replication. After two rounds, semiconservative replication would produce two DNA molecules containing only light nitrogen, and two DNA molecules containing one light strand and one heavy strand. Therefore there would

be two different densities: light and intermediate. Two rounds of dispersive replication would produce four DNA molecules, each of which would contain mostly light nitrogen and some heavy nitrogen. There would be a single density (we'll call it 'slightly heavy'). When density of the DNA was measured after two rounds, two densities were observed: light and intermediate, indicating that DNA replication is semiconservative, and not dispersive or conservative.

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Origins of Replication Although the semiconservative nature of DNA replication had been confirmed, many questions about replication remained. One of these questions was: is replication initiated at a specific site on the chromosome, or is it initiated at random sites, or even multiple sites? The answer to this question depends somewhat on the organism being considered. Bacteria, for example, have a single specific origin of replication; in other words, bacterial replication begins at the same spot on the chromosome every time. In E. coli, this site is called OriC. OriC is a 9 base-pair (bp) sequence that is repeated four times within the region. Eukaryotes also have specific sites at which replication is originated. However, because eukaryotic cells contain much more DNA than bacteria (humans have approximately 1500 times as much DNA as E.coli), there must be multiple origins of replication on each chromosome in order to replicate all of the DNA in a timely fashion. The amount of DNA replicated from a single origin is called a replicon. Other research has revealed that DNA replication proceeds bidirectionally from an origin of replication. This means that replication proceeds in opposite directions away from the origin:

Note in the diagram how each original DNA molecule branches, or forks, at the point where replication is occurring. These branch points are called replication forks. Because replication is bi-directional, two replication forks form at each origin of replication. (Some rare examples have been seen where replication is unidirectional from the origin.) The open area of the chromosome between the replication forks is called a replication bubble.

DNA Polymerase I DNA replication is catalyzed by a family of enzymes called DNA polymerases. The first of these enzymes to be discovered, DNA polymerase I, was isolated from bacteria (specifically, E. coli). Characterization of the activity of this enzyme in vitro revealed that it had certain requirements for activity. It needed 5'-triphosphate forms of the four nucleotides, and it required the presence of preexisting DNA. The DNA serves two purposes: 1) it serves as a template for the synthesis of the new DNA (the template determines the sequence of the new DNA strand, through the specificity of base pairing), and 2) it serves as a primer for DNA synthesis. It turns out that DNA polymerase I cannot initiate DNA synthesis without having a free 3'-OH to add a new nucleotide to. DNA synthesis therefore needs a primer, a preexisting piece of nucleic acid to serve as an initiator of DNA synthesis. DNA polymerase I synthesizes DNA by forming a bond between the 5' phosphate of the incoming nucleotide (the other two phosphate groups from the nucleotide triphosphate are lost) and the 3' OH group of the nucleotide at the end of the growing DNA chain. If you draw this out for yourselves, you'll realize that this means the DNA chain being synthesized grows in a 5' to 3' direction. This is an important rule to remember: DNA polymerase synthesizes DNA only in a 5' to 3' direction. In addition to its polymerase activity, DNA polymerase I has two other enzymatic activities, both of which are exonuclease activities. Exonucleases are enzymes that digest DNA (cleave phosphodiester bonds), chewing away at nucleotides from the end of the DNA chain. DNA polymerase I has 3' to 5' exonuclease activity, which degrades DNA in a direction opposite to that of synthesis. This provides the enzyme with a proofreading function: if a wrong nucleotide gets inserted into a growing chain, the enzyme can digest it out with the 3' to 5' exonuclease activity (almost like using the delete key while word processing), and insert the correct nucleotide. DNA polymerase I also has a 5' to 3' exonuclease activity, which degrades nucleic acids in the same direction as synthesis. As we'll see, this activity is used to remove primers during DNA replication. Multiple Polymerases DNA polymerase I, it turns out, is not the main enzyme involved in replicating the bacterial chromosome. When the gene encoding the enzyme was mutated in E. coli, the bacteria were still able to replicate their chromosomes. They were, however, deficient in DNA repair. This suggested that DNA polymerase I is primarily involved in DNA repair (although it does play a role in replication, as we will see), and that another yet-to-bediscovered enzyme would be responsible for replication. Eventually, two other DNA polymerases were identified, and named DNA polymerases II and III. These both had 5' to 3' polymerase activity like DNA polymerase I, and 3' to 5' exonuclease activity. Neither of these enzymes had the 5' to 3' exonuclease activity found

in DNA polymerase I. The various enzyme activities of the different polymerases are summarized in the table below. Enzyme Activity 5' to 3' polymerase 3' to 5' exonuclease 5' to 3' exonuclease DNA Polymerase DNA Polymerase DNA Polymerase I II III Yes Yes Yes Yes Yes No Yes Yes No

DNA polymerase III turns out to be the main enzyme involved in DNA replication. DNA polymerase II is a minor enzyme involved in DNA repair. DNA polymerase I is the main polymerase involved in DNA repair, and plays a specialized role in DNA replication, using its 5' to 3' exonuclease activity. The Mechanism of Prokaryotic DNA Replication As mentioned above, DNA replication in E. coli begins at OriC. It starts when the polypeptide products of the dnaA gene bind to the origin. These polypeptides cause localized strand separation. This allows a complex of the protein products of the dnaB and dnaC genes to bind. This complex acts as a helicase, which functions to unwind the DNA further. This unwinding produces the two replication forks. The unwound singlestranded region is kept single stranded through the action of single-strand binding proteins. There are other proteins that are found at the replication forks at this time, but their function is not well understood, so they will not be addressed. At this point, a primer is needed so that DNA polymerase III can begin to act. As mentioned earlier, DNA synthesis needs a primer, so how is a primer produced? An enzyme called primase serves this purpose, by synthesizing a short stretch of RNA (generally from 5 to 15 nucleotides in length). RNA synthesis does not require a primer, so primase (which is a type of enzyme called an RNA polymerase) is able to synthesize a short primer where needed. Once this primer is made, DNA synthesis can begin, extending the polynucleotide chain originating with the RNA primer. Priming and synthesis occurs on both strands in the helicase complex moves along the parental DNA, shifting the replication fork, and allowing synthesis to continue.

This leads to another problem that has to be solved. As more DNA unwinds, and the replication fork moves along, synthesis of one strand (the lower strand in the diagram) can just continue, following the movement of the replication fork. The other strand being synthesized, however, cannot do this. As the replication fork moves along, it leaves a gap behind, as shown in panel B of the figure. To compensate for this, a second RNA primer must be synthesized a bit behind the first one, and DNA synthesized until it reaches the first primer (this is shown in panel C). You can easily imagine that as the replication fork progresses a bit further, this process will have to be repeated. Therefore, at each replication fork, the synthesis of one new DNA strand (the lower one in the figure) is continuous, while synthesis of the other strand must be accomplished in small increments, short stretch after short stretch; this type of synthesis is termed discontinuous. The strand of DNA that is synthesized continuously is called the leading strand, and the strand that is synthesized discontinuously is called the lagging strand. The small fragments of DNA making up the lagging strand are named Okazaki fragments, after the researcher who discovered them. Okazaki fragments are typically about 1000 to 2000 nucleotides each. Discontinuous replication solves one problem but still leaves one matter unsettled: the lagging strand will be composed of individual, unjoined fragments of DNA and RNA. This is where DNA polymerase I comes into play. DNA polymerase I uses its 5' to 3' exonuclease activity to digest away the primer RNA, and replaces the primer with DNA by extending the strand from the adjacent Okazaki fragment. At this point all that is left to be done is to physically join the Okazaki fragments. This is accomplished by an enzyme known as DNA ligase. DNA ligase is able to join the 5' end of one DNA strand to the 3' end of another DNA strand. Eukaryotic DNA Replication Synthesis of DNA in eukaryotes is less well understood, but the process appears to be basically the same as in prokaryotes, with a few notable exceptions. For one thing, eukaryotic DNA is complexed with histones to form chromatin. Every round of replication, therefore, requires that the histones be removed, then replaced after replication is complete. This requirement understandably slows the whole replication process down.

Eukaryotic cells are also much more complex than prokaryotes, because they contain organelles such as mitochondria and chloroplasts (in plants) that contain their own DNA, which must also be replicated. Eukaryotic cells therefore have more than three DNA polymerases; there have been five DNA polymerases identified so far. Eukaryotes have another difference: eukaryotic chromosomes are linear, rather than circular as in prokaryotes.

DNA Replication: Summary of Key Points

DNA replication is semiconservative, with each existing strand serving as a template for the synthesis of a new strand. Replication begins at specific locations called origins of replication. Replication requires a primer , and proceeds bidirectionally from the point of origin, creating an expanding replication bubble. On one strand (the leading strand), synthesis is continuous; on the other strand (the lagging strand) synthesis is discontinuous, producing a series of Okazaki fragments that must be ligated together.

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