Question

1. Discuss different models for DNA replication that were considered by early researchers. Explain why the current model for DNA replication was accepted following experiments by Meselson and Stahl. Describe this model in detail and interpret the significance of helicases, gyrases, RNA primer, DNA polymerase, Okazaki fragments, and DNA ligase.

Answer 1

There were three models of replication possible from such a scheme: conservative, semi-conservative, and dispersive. In conservative replication, the two original DNA strands, known as the parental strands, would re-basepair with each other after being used as templates to synthesize new strands; and the two newly-synthesized strands, known as the daughter strands, would also basepair with each other; one of the two DNA molecules after replication would be “all-old” and the other would be “all-new”. In semi-conservative replication, each of the two parental DNA strands would act as a template for new DNA strands to be synthesized, but after replication, each parental DNA strand would basepair with the complementary newly-synthesized strand just synthesized, and both double-stranded DNAs would include one parental or “old” strand and one daughter or “new” strand. In dispersive replication, after replication both copies of the new DNAs would somehow have alternating segments of parental DNA and newly-synthesized DNA on each of their two strands.

Meselson and Stahl

Meselson and Stahl were interested in understanding how DNA replicates. They grew E. coli for several generations in a medium containing a “heavy” isotope of nitrogen (15N) that is incorporated into nitrogenous bases and, eventually, into the DNA. The E. coliculture was then shifted into medium containing the common “light” isotope of nitrogen (14N) and allowed to grow for one generation. The cells were harvested and the DNA was isolated. The DNA was centrifuged at high speeds in an ultracentrifuge in a tube in which a cesium chloride density gradient had been established. Some cells were allowed to grow for one more life cycle in 14N and spun again.

During the density gradient ultracentrifugation, the DNA was loaded into a gradient (Meselson and Stahl used a gradient of cesium chloride salt, although other materials such as sucrose can also be used to create a gradient) and spun at high speeds of 50,000 to 60,000 rpm. In the ultracentrifuge tube, the cesium chloride salt created a density gradient, with the cesium chloride solution being more dense the farther down the tube you went. Under these circumstances, during the spin the DNA was pulled down the ultracentrifuge tube by centrifugal force until it arrived at the spot in the salt gradient where the DNA molecules’ density matched that of the surrounding salt solution. At the point, the molecules stopped sedimenting and formed a stable band. By looking at the relative positions of bands of molecules run in the same gradients, you can determine the relative densities of different molecules. The molecules that form the lowest bands have the highest densities.

DNA from cells grown exclusively in 15N produced a lower band than DNA from cells grown exclusively in 14N. So DNA grown in 15N had a higher density, as would be expected of a molecule with a heavier isotope of nitrogen incorporated into its nitrogenous bases. Meselson and Stahl noted that after one generation of growth in 14N (after cells had been shifted from 15N), the DNA molecules produced only single band intermediate in position in between DNA of cells grown exclusively in 15N and DNA of cells grown exclusively in 14N. This suggested either a semi-conservative or dispersive mode of replication. Conservative replication would have resulted in two bands; one representing the parental DNA still with exclusively 15N in its nitrogenous bases and the other representing the daughter DNA with exclusively 14N in its nitrogenous bases. The single band actually seen indicated that all the DNA molecules contained equal amounts of both 15N and 14N.

The DNA harvested from cells grown for two generations in 14N formed two bands: one DNA band was at the intermediate position between 15N and 14N and the other corresponded to the band of exclusively 14N DNA. These results could only be explained if DNA replicates in a semi-conservative manner. Dispersive replication would have resulted in exclusively a single band in each new generation, with the band slowly moving up closer to the height of the 14N DNA band. Therefore, dispersive replication could also be ruled out.

Meselson and Stahl’s results established that during DNA replication, each of the two strands that make up the double helix serves as a template from which new strands are synthesized. The new strand will be complementary to the parental or “old” strand and the new strand will remain basepaired to the old strand. So each “daughter” DNA actually consists of one “old” DNA strand and one newly-synthesized strand. When two daughter DNA copies are formed, they have the identical sequences to one another and identical sequences to the original parental DNA, and the two daughter DNAs are divided equally into the two daughter cells, producing daughter cells that are genetically identical to one another and genetically identical to the parent cell.

ecause eukaryotic genomes are quite complex, DNA replication is a very complicated process that involves several enzymes and other proteins. It occurs in three main stages: initiation, elongation, and termination.

Initiation

Eukaryotic DNA is bound to proteins known as histones to form structures called nucleosomes. During initiation, the DNA is made accessible to the proteins and enzymes involved in the replication process. There are specific chromosomal locations called origins of replication where replication begins. In some eukaryotes, like yeast, these locations are defined by having a specific sequence of basepairs to which the replication initiation proteins bind. In other eukaryotes, like humans, there does not appear to be a consensus sequence for their origins of replication. Instead, the replication initiation proteins might identify and bind to specific modifications to the nucleosomes in the origin region.

Certain proteins recognize and bind to the origin of replication and then allow the other proteins necessary for DNA replication to bind the same region. The first proteins to bind the DNA are said to “recruit” the other proteins. Two copies of an enzyme called helicase are among the proteins recruited to the origin. Each helicase unwinds and separates the DNA helix into single-stranded DNA. As the DNA opens up, Y-shaped structures called replication forks are formed. Because two helicases bind, two replication forks are formed at the origin of replication; these are extended in both directions as replication proceeds creating a replication bubble. There are multiple origins of replication on the eukaryotic chromosome which allow replication to occur simultaneously in hundreds to thousands of locations along each chromosome.

Elongation

During elongation, an enzyme called DNA polymerase adds DNA nucleotides to the 3? end of the newly synthesized polynucleotide strand. The template strand specifies which of the four DNA nucleotides (A, T, C, or G) is added at each position along the new chain. Only the nucleotide complementary to the template nucleotide at that position is added to the new strand.

DNA polymerase contains a groove that allows it to bind to a single-stranded template DNA and travel one nucleotide at at time. For example, when DNA polymerase meets an adenosine nucleotide on the template strand, it adds a thymidine to the 3? end of the newly synthesized strand, and then moves to the next nucleotide on the template strand. This process will continue until the DNA polymerase reaches the end of the template strand.

DNA polymerase cannot initiate new strand synthesis; it only adds new nucleotides at the 3? end of an existing strand. All newly synthesized polynucleotide strands must be initiated by a specialized RNA polymerase called primase. Primase initiates polynucleotide synthesis and by creating a short RNA polynucleotide strand complementary to template DNA strand. This short stretch of RNA nucleotides is called the primer. Once RNA primer has been synthesized at the template DNA, primase exits, and DNA polymerase extends the new strand with nucleotides complementary to the template DNA.

Eventually, the RNA nucleotides in the primer are removed and replaced with DNA nucleotides. Once DNA replication is finished, the daughter molecules are made entirely of continuous DNA nucleotides, with no RNA portions.

Termination

Eukaryotic chromosomes have multiple origins of replication, which initiate replication almost simultaneously. Each origin of replication forms a bubble of duplicated DNA on either side of the origin of replication. Eventually, the leading strand of one replication bubble reaches the lagging strand of another bubble, and the lagging strand will reach the 5? end of the previous Okazaki fragment in the same bubble.

DNA polymerase halts when it reaches a section of DNA template that has already been replicated. However, DNA polymerase cannot catalyze the formation of a phosphodiester bond between the two segments of the new DNA strand, and it drops off. These unattached sections of the sugar-phosphate backbone in an otherwise full-replicated DNA strand are called nicks.

Once all the template nucleotides have been replicated, the replication process is not yet over. RNA primers need to be replaced with DNA, and nicks in the sugar-phosphate backbone need to be connected.