CHAPTER 4

DNA replication:

DNA is replicated in a semiconservative manner during the S phase of the cell cycle. Meselson and Stahl demonstrated the semiconservative nature using density gradient centrifugation techniques. See foundations of Genetics 4-1.

DNA replication occurs at origin points where DNA is unwound and replication bubbles form, each containing two replication forks. Each fork is being operated on simultaneously. Prokaryotes have a single origin point, eukaryotes have many. The DNA must be unwound prior to being processed for replication. This is controlled by a helicase. The two strands are stabilized such that they do not reassemble due to the attachment of single stranded binding proteins (SSBP). The polymerization of the new DNA molecule occurs in a 5' to 3' direction (the DNA is read 3' to 5') and is under the control of DNA polymerase. In prokaryotes, the majority of DNA is synthesized by DNA polymerase III. DNA cannot begin synthesis of a DNA strand without the availability of a 3' end of an existing sugar phosphate backbone. This problem is circumvented by first synthesizing an RNA primer to provide an available 3' end for the DNA polymerase to use. The primer is created using primase (an RNA polymerase) and is 5-15 nucleotides long. As the bubble enlarges, kinks in the DNA are eliminated by gyrases (topisomerases). Due to the antiparallel nature of the DNA, one strand can effectively be synthesized continuously (the leading strand) while the other must be synthesized discontinuously (the lagging strand) in Okazaki fragments. On the 5' end of each Okazaki fragment is an RNA primer which is eventually digested and replaced with DNA by DNA polymerase I  ( the only DNA polymerase with 5' to 3' exonuclease activity) that attaches to the 3' end of the upstream Okazaki fragment and extends the DNA replacement. The DNA of the new strand is eventually joined together by ligase. (Figs. 4-5, 4-6, 4-7, and 4-8). DNA polymerase II is involved with DNA repair.

Eukaryotes have a different set of DNA polymerases: alpha (involved in priming DNA for replication, beta and epsilon (involved in DNA repair), gamma (synthesizes mitochondrial DNA) and delta (involved in replicating most nuclear DNA). The RNA primer is not removed by a DNA polymerase in eukaryotes, but instead by a pair of nucleases. HI endonuclease recognizes the union of RNA and DNA strands and nicks the backbone at such locations FEN1 is an RNA exonuclease that then digests the RNA primer which in turn is replaced by DNA polymerase alpha.

Cell division:

In prokaryotes, DNA replication and cell division occur via binary fission.

In eukaryotes, cell division involves nuclear divisions (Mitosis or Meiosis) followed by cytokinesis. DNA replication occurs during the S phase of the cell cycle generating genetically identical sister chromatids united by a common centromere. Mitosis generates two genetically identical nuclei.Meiosis generates four nuclei that each have half the number of chromosomes that the original nucleus had, but one of each kind of chromosome.

Mitosis:

Prophase- Nuclear envelope disassociates. DNA condenses and chromosomes become visible, microtubules attach to kinetochores on centromeres and direct their movement to the equatorial plane.

Metaphase- Centromeres line up along the equatorial plane such that one member of each sister chromatid pair is oriented toward one pole and the other sister chromatid toward the other pole.

Anaphase- Sister chromatids separate from each other (becoming chromosomes) and migrate to opposite poles directed by microtubules.

Telophase- Two nuclear envelopes reassemble, DNA decondenses.

Meisosis:

A sequence of two divisions, the first (the reduction division) reduces the number of chromosomes in half, the second (the equational division) is like a mitotic division.

Prophase I- Nuclear envelope disassociates, Chromosomes condense, homologous chromosomes pair in synapsis. Synaptomenal complex forms, Chiasmata become visible, crossing over occurs. Chromosomes move due to microtubules.

Metaphase I- Homologous chromosomes line up on opposite sides of the equatorial plane. The arrangement of each homologous pair is independent of the other nonhomologous pairs.

Anaphase I- Homologous chromosomes segregate and move to different poles.

Telophase I- Variable across species

Prophase II- similar to prophase of mitosis

Metaphase II- similar to metaphase of mitosis

Anaphase II- similar to anaphase of metaphase

Telophase II- similar to telophase of mitosis

Meiosis immediately precedes gamete formation in animals.

Some mechanical differences arise between replication of DNA in a circular configuration versus replication of linear strands of DNA. First there appears to be only a single origin of replication in prokaryotes. In bacteria, this leads to a replication bubble forming and expanding such that the replicating DNA takes on the appearance of the Greek letter theta (Fig. 4-9). Often viral DNA replicates according to a model referred to as the rolling circle (Fig. 4-10). This involves one strand of the DNA being clipped by an endonuclease, and then the two sugar phosphate strands separating as shown in the figure. The strand that was not nicked serves as a template for a newly synthesized strand that begins on the 3' end of the nicked strand. This process results in multiple copies being made of the complement to the intact strand all of which remain joined together and later serve themselves as a single stranded template for the formation of Okazaki fragments. The Okazaki fragments are processed with DNA polymerase I and ligases to form a continuous double stranded DNA molecule that contains multiple repeats of the original genome. This is called a concatamer. The concatamer is then processed with endonucleases to cleave it into separate genomes identical to the original genome.

 A problem arises during replication of the ends of linear DNA molecules. At the 3' end of the template for the lagging strand an RNA primer is synthesized and extended by a DNA polymerase. When the RNA is removed by the H1 and FEN1 it can not be replaced by DNA polymerase activity because there is no DNA sugar phosphate strand available to extend. Therefore, the end of the DNA molecule has a short single stranded length. Each subsequent replication, would lead to a shorter and shorter DNA molecule. This would continue until the telomere shortened sufficiently to disrupt normal cellular activity. This may put an upper limit on the number of divisions possible for a cell line. In cell cultures, it has been noted that the maximum number of divisions is about 50 (this is referred to as the Hayflick Number). Some cells do not seem to be impeded by this process (e.g., tumor cells, stem cells, etc.) and these cells produce an enzyme (telomerase) that prevents excessive shortening of the DNA. Telomerase is a combination of RNA and protein that attaches to the end of the DNA and extends the 3' end of the lagging strand template with repeats of the same six nucleotides. These six nucleotides are complementary to six nucleotides on the RNA molecule contained in the telomerase.