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Biology Chapter 14.docx

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McMaster University
Lovaye Kajiura

Biology Chapter 14: DNA and the Gene: Synthesis and Repair Bernard Ho November 13, 2010 DNA as the Hereditary Material − Frederick Griffith reported the discovery of a mysterious phenomenon involving hereditary traits − He referred to this phenomenon as transformation − His transformation experiments appeared to isolate the hereditary material − Griffith was doing experiments in an attempt to develop a vaccine against the Streptococcus pneumoniae bacterium − He worked with strains of the bacterium that infect mice − A strain is a population of genetically identical individuals − As is the case with strains that affect humans, the strains that affect mice vary in their virulence, their ability to cause disease and death − A medium is a liquid or solid that is suitable for growing cells − On a solid medium, cells from the non-virulent strain form colonies that look rough (R) and cells fro the virulent strain form colonies that look smooth (S) − To understand how the strains interact, Griffith designed four experimental treatments − In the first treatment, he injected mice with cells of the R strain and as expected, the mice lived − In the second treatment, he injected mice with cells of the S strain and as expected, these mice died of pneumonia − In the third treatment, Griffith killed cells of the S strain by heating them and then injecting them into the mice o These mice lived, which showed that dead S cells do not cause disease − In the final treatment, Griffith injected mice with heat-killed S cells and live R cells o Unexpectedly, these mice died o Autopsies confirmed pneumonia as the cause of death o When he isolated and grew the bacteria from these dead mice, he found S cells, not R cells o He proposed that something from the heat-killed S cells had transformed the non-virulent R cells o Something had changed the appearance and behaviour of the R cells from R-like to S-like o Because this “something” appeared in the growing population of cells that Griffith isolated from dead mice, it had been passed on to the offspring of the transformed cells o Clearly a hereditary factor − Due to the confirmation of the Chromosome Theory, it was clear that Griffith’s transforming factor had to consist of DNA or protein o Initially, most biologist backed the hypothesis that genes are made of proteins − The Avery et al. experiment o To determine whether protein, RNA or DNA was responsible for transformation, they grew quantities of S cells in culture o They killed the cultured cells with heat, broke them open to create a cell extract and then used chemical treatments to remove the lipids and carbohydrates from the extracts o These steps left the mixture containing protein, RNA and DNA from the S cells o The researchers divided the sample into three treatments and used different enzymes to destroy a specific macromolecule in each o One sample was treated with proteases, which destroy protein o Another sample was treated with ribonuclease, which breaks apart RNA o The other sample was treated wit DNAase, an enzyme that cuts up DNA o When small quantities of the three resulting solutions were added to cultures containing R cells, S cells appeared in all of the cultures that still contained S-cell DNA, so no S cells appeared in the sample that lacked DNA o The biologists concluded that DNA must be the transforming factor − The Hershey-Chase Experiment o Alfred Hershey and Martha Chase took up the question of whether genes are made of protein or DNA by studying how a virus called T2 infects the bacterium Escherichia coli o They knew that T2 infections begin when the virus attaches to the cell wall of E. coli and injects its genes into the cell’s interior o These genes then direct the production of a new generation of virus particles inside the infected cell, which acts as a host for the parasitic virus o During the infection the protein coat of the original parent virus is left behind, still attached to the exterior of the host cell as a “ghost” o They also knew that T2 is made up of almost entirely protein and DNA o Their strategy for determining which part of the virus enters the cell and acts as the hereditary material was based on two facts  Protein present in T2 contains sulphur, but not phosphorus  DNA contains phosphorus, but not sulphur o The researchers began their work by growing viruses in the presence of either the radioactive isotope of sulphur ( S) or the radioactive isotope of 32 phosphorus ( P) o Because these molecules were incorporated into newly synthesized proteins and DNA, this step produced a population of viruses with radioactive proteins and a population with radioactive DNA o Then, they let each set of radioactive viruses infect E. coli o If genes consist of DNA, then the radioactive protein should be found in the empty, or “ghost” protein coats outside the infected host cell, while the radioactive DNA should be located inside the cells o But if the genes consist of proteins, the opposite is true o To test these predictions, they shook the ghosts of the cells using a kitchen blender o When the researchers spun the samples in a centrifuge, the ghosts stayed in solution, while the cells formed a pellet at the bottom of the centrifuge tube o As predicted by the DNA hypothesis, the biologists found that almost all of the radioactive protein was in the ghosts, while all the radioactive DNA was inside the host cells o Because the injected component of the virus directs the production of a new generation of virus particles, it is this component that represents the virus’ genes Testing Early Hypothesis about DNA Synthesis − Recall that DNA is a long, linear polymer made up of monomers called deoxyribonucleotides, which consist of a deoxyribose molecule, a phosphate group and a nitrogenous base − Deoxyribonucleotides link together into a polymer when a phosphodiester bond forms between a hydroxyl group on the 3’ carbon of deoxyribose and the phosphate group attached to the 5’ carbon of deoxyribose − The primary structure of DNA has two major components o A backbone made up of sugar and phosphate groups of deoxyribonucleotides o A series of nitrogenous bases that project inward from the backbone − A strand of DNA has directionality, one end has an exposed hydroxyl group on the 3’ carbon, while the other has an exposed phosphate group on the 5’ carbon − The double stranded molecule that results from the anti-parallel strands is called a double helix − Watson and Crick suggested that the existing strands of DNA served as a template for the production of new strands, with bases being added to the new strands according to complementary base pairing − There were three other hypotheses about how the old and new strands might interact during replication o If old strands of DNA separated, they could then be used as a template for the synthesis of a new daughter strand  This hypothesis is called semi-conservative replication because each new daughter molecule would consist of one old strand and one new strand o If bases temporarily turned outward so that complementary strands no longer faced each other, they could serve as a template for the synthesis of an entirely new double helix at once  This hypothesis is called conservative replication  Results in the intact parental strands and a daughter molecule consisting entirely of newly synthesized strands o If the parent helix was cut and unwound in short sections before being copied and put back together, then new and old strands would intermingle, old sections of DNA would be interspersed with new DNA down the length of each daughter molecule  This hypothesis is called dispersive replication − The Meselson-Stahl Experiment o They realized that if they could tag parental and daughter strands of DNA in a way that would make them distinguishable from each other, they could determine whether replication was conservative, semi-conservative or dispersive o They decided to work with E. coli o Like all organisms, bacterial cells copy their entire complement of DNA (genome) before every cell division o To distinguish parental strands of DNA from daughter strands when E. coli replicates, they grew cells for many generations in the presence of 15 14 isotopes of nitrogen ( N and N) o The difference in mass of the two isotopes ( N is heavier), which crates a difference in density of 1N-containing and 1N-containing DNA, was the key to the experiment o The biologists reasoned that if different nitrogen isotopes were available in the growth medium when parental and daughter strands of DNA were produced, then the two types of strands should behave differently during centrifugation o When intact, double stranded DNA molecules are added to a solution that forms a gradient from low to high density during centrifugation, DNA 15 strands that contain N should form a band in the lower-density part of the centrifuge tube o In contrast, DNA strands that contain 15N should form a band in the higher-density part of the centrifuge tube o Because the highest density solution is a the bottom of the tube, DNA that contains N should be found lower in the tube than DNA containing N 14 o In this way, DNA strands containing the two isotopes should form separate bands o The researchers began by growing the bacterial cells with nutrients that contained only N15 o They purified DNA from a sample of these cells and transferred the rest of 14 the culture to a growth medium containing only the N isotope o After enough time had elapsed for these experimental cells to divide once, they removed a sample and isolated the DNA o After the remainder of the culture had divided again, they removed another sample and purified the DNA o If replication is conservative, then the daughter cells should have double- 14 15 stranded DNA with either N or N, but not both  As a result, two distinct DNA bands should form in the centrifuge tube, one high density band and one low density band o If replication is semi-conservative or dispersive, then all of the experimental DNA should contain an equal mix of both isotopes after one generation and one intermediate-density band should form in the centrifuge tube  But after two generations, half of the daughter cells should contain only 1N if replication is semi-conservative, meaning a second, lower density band should appear in the centrifuge tube  But the dispersive model predicts that there will be just one bad at intermediate density o Results  After one generation, the density of the DNA molecules was intermediate  Suggested that conservative replication was wrong  After two generations, a lower-density bad appeared in addition to the intermediate-density band  This result offered strong support for the hypothesis that DNA replication is semi-conservative A Comprehensive Model for DNA Synthesis − DNA polymerase polymerizes deoxyribonucleotides to DNA − This protein catalyzes DNA synthesis − However, DNA polymerases can only work in one direction − They can add deoxyribonucleotides to only the 3’ end of the growing DNA chain − As a result, DNA synthesis always proceeds in the 5’  3’ direction − How replication gets started o A “bubble” forms in a chromosome when DNA is actively being synthesized o Bacteria chromosomes have a single location where the replication process begins and thus a single bubble forms o Initially, the replication bubble forms at a specific sequence of bases called the origin of replication o Replication bubbles grow as DNA replication proceeds because synthesis is bidirectional (occurs in both directions)(but always 5’  3’ because strands are anti-parallel) o Eukaryotes have bidirectional replication, but they have multiple sites along each chromosome where DNA synthesis begins and thus multiple replication bubbles o A specific suite of proteins is responsible for recognizing sites where replication begins and opening the double helix at those points o These proteins are activated by the proteins responsible for initiating S phase in the cell cycle o Once a replication bubble opens, a different suite of enzymes takes over and initiates replication o A replication fork is a Y-shaped region where the parent DNA double helix is split into two single strands, which are then copied − How helix is opened and stabilized o Enzyme helicase catalyzes the breaking of hydrogen bonds between deoxyribonucleotides, causing the two strands of DNA to separate o Proteins called single-strand DNA binding-proteins (SSBPs) attach to the separate strands and prevent them from snapping back into a double helix o In combination, the helicase and single-strand DNA-binding proteins open up the double helix and make both strands available for copying o The unzipping process that occurs at the replication fork creates tension farther down the helix o Ex. Rope  Imagine what would happen if you started to pull apart the twisted strands of a rope  The untwisting movements at one end would force the intact section to rotate in response  If the intact end of the rope were fixed in place, it would eventually begin to coil on itself and kink in response to the twisting forces (supercoiling) o This does not happen in DNA because the twisting stress induced by helicase is relieved by proteins called topoisomerases o Topoisomerase is an enzyme that cuts and rejoins the DNA downstream of the replication fork − How the leading strand is synthesized o DNA polymerase III works only in the 5’ 3’ direction and to start synthesis, it requires both a 3’ end and a single-stranded template o The single stranded template dictates which deoxyribonucleotides should be added next, while the primer, which consists of a few nucleotides bonded to the template, provides a free 3’ hydroxyl group that can
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