GEN 3022 1st Edition Lecture 16Outline of Last Lecture I. Structure of bacterial chromosomesa. Locationb. Overall shape/structurei. Loop domainsii. DNA supercoilingII. Organization of Eukaryotic chromosomesa. Chromosome replication and segregationi. Three types of DNA sequencesb. Compaction of DNAi. Process: interphase and metaphaseii. Heterochromatiniii. EuchromatinIII. Nucleosomesa. Definitionb. Componentsi. Histone proteinsii. DNAOutline of Current LectureI. Overview of DNA replicationThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.a. Replication patternsi. Antiparallelii. Chargraff’s ruleiii. Semiconservativeb. SummaryII. Proposed models of DNA replicationa. Conservative modelb. Semiconservative modelc. Dispersive modelIII. Experimenta. E. coli growthb. Light and half-heavy DNA typesIV. Origin of bacterial DNA replicationa. Origin of replication (oriC)b. Patterns of bacterial DNA replicationV. Synthesis of new DNA strandsa. DNA helicaseb. Topoisomerase II (DNA gyrase)c. Primased. DNA Polymerasese. LigaseVI. Synthesisa. Leading strands b. Lagging strandsc. DNA polymerase I actiond. DNA ligase actionVII. Fidelity mechanismsa. High fidelity in DNA replicationsi. Three reasonsb. Proofreading activity of DNA polymeraseVIII. Eukaryotic genomesIX. Telomeres and DNA replicationa. Telomeric sequencesb. Telomerase c. Telomere length and cancerCurrent LectureI. Overview of DNA replicationa. Replication patternsi. Antiparallel: one strand runs 5’ to 3’ and the other 3’ to 5’ ii. Chargraff’s rule: A+T = C+Giii. Semiconservative: one parent strand and one daughter strandb. Summaryi. Two complementary strands of DNA separateii. Each serves as a template strand for the synthesis of new complementary (daughter) DNA strandsII. Proposed models of DNA replicationa. Conservative model: both parental strands stay together after DNA replicationb. Semiconservative model: the double-stranded DNA contains one parental and one daughter strand following replicationc. Dispersive model: parental and daughter DNA segments are interspersed in both strands following replicationIII. Experimenta. E. coli growth: Meselson and Franklin Stahl investigated DNA replication using E. coli i. E. coli was grown in the presence of a heavy isotope of nitrogen so that the population of cells all had heavy labeled DNAii. E. coli was switched to a medium with only light isotope of nitrogeniii. Density of resulting generations was analyzed to determine the pattern ofreplicationb. Light and half-heavy DNA typesi. The first generation’s DNA types were consistent with the dispersive model and semiconservative model, but the second generation’s types were only consistent with the semi-conservative modelIV. Origin of bacterial DNA replicationa. Origin of replication (oriC): since bacterial chromosomes are circular, there is onlyone origin of replication (called the origin of Chromosomal replication: oriC)b. Patterns of bacterial DNA replication: synthesis of DNA proceeds bidirectionally around the chromosome until the replication forks meet and replication is terminatedV. Synthesis of new DNA strandsa. DNA helicase: responsible for separating the two template strands into a replication forkb. Topoisomerase II (DNA gyrase): unwinds negative supercoiling of DNA so that it can be separated for replicationc. Primase: creates RNA primer segments that covalently link to the template strand and are later removed by DNA polymerase Id. DNA Polymerases: responsible for DNA synthesis, work in the 5’ to 3’ direction only and cannot initiate transcription. DNA polymerase III is responsible for the most synthesis. e. Ligase: covalently links Okazaki fragments of lagging strand by catalyzing formation of the phosphodiester bondVI. Synthesisa. Leading strand: strand that gets synthesized continuously (using one RNA primer)from 5’ to 3’ towards replication forkb. Lagging strand: strand that gets synthesized in fragments from 5’ to 3’ away fromthe replication forkc. DNA polymerase I action: removes RNA primer and fills in the gaps with newly synthesized DNAd. DNA ligase action: covalently links Okazaki fragments after DNA polymerase I fills in the “gaps” that are created by the removal of RNA primerVII. Fidelity mechanismsa. High fidelity in DNA replications (mistakes during DNA replication are extremely rare; DNA polymerase III makes only one mistake per every 108 bases made)i. Three reasons: stability of base pairing, structure of DNA polymerase active site, proofreading function of DNA polymeraseb. Proofreading activity of DNA polymerase: DNA polymerases can identify a mismatched nucleotide and remove it from the daughter strand. The enzyme uses a 3’ to 5’ exonuclease activity to digest the newly made strand until the mismatched nucleotide is removed.VIII. Eukaryotic genomesa. Unlike bacterial genomes, eukaryotic chromosomes are long and linear (as opposed to circular)b. As a result they also have many origins of replication (about every 100,000 base pairs)IX. Telomeres and DNA replicationa. Telomeric sequencesi. Sequences at the end of a chromosome that codes for the stop of replicationii. Typically consist of moderately repetitive tandem arrays with a 3’ overhang that is 12-16 nucleotides in length. These nucleotides are generally many guanine and thymine.b. Telomerase i. Since DNA polymerases can only work in the 5’ to 3’ direction and cannot initiate DNA synthesis, a special enzyme (telomerase) is needed to complete replication. ii. There is not enough DNA at the end of a strand to allow for RNA primer to attach, which prevents DNA replication from completion. iii. Telomerase builds on to the end of a DNA strand to allow for RNA primer to bind and for replication to finish. c. Telomere length and canceri. Telomere DNa is about 8,000 at birth and can shorten to 1,500 bp in an elderly person.ii. Telomeres shorten in actively dividing cells, which makes some cells senescent (loss of ability to divide). Insertion of highly active telomeres can block senescence. iii. Cancer cells commonly carry mutations increasing activity of telomerase, which prevents telomere shortening and senescence. May be a target for anti-cancer drug