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UW-Milwaukee BIOSCI 150 - DNA

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Outline of Last LectureOutline of Current LectureCurrent LectureDNAHistoryComposistionReplication Of DNAReplication Factors:DNA Replication In ProkaryotesReplication In EukaryotesRolling Circle ReplicationBIO SCI 150 1st Edition Lecture 9Outline of Last Lecture 1. Cell Divisiona. Key terms b. Cell Cyclei. Mitosis1. Components2. Stagesii. Meiosis1. Gametes2. Stagesa. Meiosis 1b. Meiosis 2iii. ReproductionOutline of Current Lecture 1. DNAa. Historyb. Compositionc. Replicationi. Factorsii. In prokaryotesiii. In eukaryotesd. Rolling Circle ReplicationCurrent LectureDNAHistoryGriffith Transformation (1928)- Use two strains of bacterium Streptococcus pneumoniae- Genetic information is not destroyed when organism is killed, but can“transform”another living cell and give it new traits.Avery, McCarty and MacLeod Identify Transforming Substance (1944)- Isolated chemicals from heat-killed bacteria and showed thatdestroying DNAstops transformation, but destroying RNA or proteindo not.Hershey and Chase Virus DNA (1952)- DNA carries the genetic information, or transforming agentWatson and Crick DNA Structure is Double Helix (1954)- Used chemical, physical and X-ray data to helix determine structureMeselson and Stahl DNA Replication is Semiconservative (1958)- Cells were grown in heavy nitrogen, then transferred to mediacontaining light nitrogen. Using centrifugation to separate heavy andlight molecules they showedthat after one generation each DNAmolecule contained one heavy (old) strand and one light (newlymade) strand.ComposistionDNA is composed of nucleotides joined by a phospho-diester linkage thatlinks the 3’ carbon of one nucleotide to the 5’ carbon of the next nucleotide via the oxygen atoms ofa phosphate group - DNA double helix is composed of two strands lying antiparallel. - Because the two strands are not in the same orientation the helix has two grooves of different size termed the major andminor groove. The two strands areheld together by hydrogen bonds.- Nucleotide contains nitrogenous base, deoxyribose and a phosphate group.Replication Of DNA- Types of replication:o semiconservative each progeny molecule contains one of the parental strands.o conservative two parental strands remain together and the daughter molecule has two new strands.o dispersive the parental DNA is dispersed through out the newmolecule- Meselson and Stahl demonstrated that replication was semiconservativeo Old strands are used as the template for the new strands.o Each new double strand has one old and one new strand.- Replication occurs by adding a 5’ triphosphate to the 3’ OH of an existing DNA strand to base pair with the template strand.- Replication requires a starting point called the origin of replication- Replication requires a 3’OH on an existing DNA strand- Replication requires a template strandReplication Factors:1. Initiator protein: binds to the origin of replication, directs the helicases tothe DNA.2. Helicases: open up and unwind the DNA at the replication fork.3. Gyrases (topoisomerase II): removes the positive supercoils induced by the unwinding of the DNA at the replication fork. This is done by introducingnegative supercoils.4. SSBPs: (single stranded binding proteins) keep loop open by preventing the single stranded DNA from folding back on itself or reforming with the otherstrand.5. Primase: an RNA polymerase which makes the RNA primer required by the DNA polymerase III to begin making DNA.6. DNA Polymerase III: (the major replication enzyme) adds nucleotides onto the 3’ end of an existing DNA chain or RNA primer.7. DNA Polymerase I: the cut and paste enzyme. Also called the repair enzyme. Removes the RNA primers and replaces them with DNA.8. Nucleases: break bonds between nucleotides endonuclease (cuts within a chain) and exonuclease (cuts off terminal nucleotide).9. Ligase: joins nucleotide chains together.10. Replicon: the chromosomal segment between replication initiation sitesDNA Replication In Prokaryotes1. Replication bubble forms on circular chromosome when the Initiatorproteins open the origin of replication.2. Helicases: open up and unwind the DNA at the replication fork.3. Primase makes RNA primer with free 3’ OH for leading strand4. DNA polymerase III begins making DNA, adding nucleotides to 3’ end5. DNA polymerase III elongates leading strand, continuous replication6. Primase makes RNA primer for the lagging strand7. DNA Polymerase III adds nucleotides to the 3’ end of the lagging strand, discontinuous replication8. Lagging strand is replicated in short fragments called Okazaki fragments9. DNA Polymerase I Removes RNA primers and replaces them with DNA.10. Ligase joins Okazaki fragments togetherReplication is bi-directional, in that replication forks move out in both directions from the origin of replication. At each fork the 3’ ends of both leading and laggingstrands are elongated. The polymerase molecules move together as a replisome.The lagging strand is folded over and is replicated slightly after the leading strand.Replication In EukaryotesSimilar to prokaryotes- RNA primer required- Similar enzymes required, polymerases, nucleases, ligases- synthesis is 5’ to 3’- leading and lagging strands are presentDifferences- DNA replication is in S phase- Different polymeraseso Alpha and delta: replication of DNAo Beta: DNA repairo Gamma: found in the mitochondriao multiple origins of replication are present on each chromosomeo DNA is incorporated immediately with new histone proteinsRolling Circle Replication- Occurs in some viruses, bacterial mating, and oocytes to make multiple copies.- One strand of double-stranded circular DNA remains intact and serves as “rolling” template. Newly made strand serves as template for complementary


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