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BIOLOGY 2CHAPTER 15 NOTES: DNA AND THE GENE: SYNTHESIS AND REPAIR15.1 What are genes made of?- DNA or protein? Originally thought proteins b/c limitless variation in structure/functiono Avery showed early on that DNA could be genetic materialo Hershey-Chase experiment: studied how T2 virus (made of primarily DNA and protein) infected E. coli Proteins have sulfur, DNA has phosphorus Grow viruses with radioactive labels on either S or P and infect E. coli cells, then locate where radioactivity is found Centrifuge to separate empty capsids from bacterial cells Radioactive protein was in capsids outside cells, radioactive DNA inside – DNA is hereditary material- Watson and Crick  DNA runs antiparallel and form a double helix following complimentary base pairing15.2 Testing early hypotheses about DNA synthesis- W & C  base pairing serves as template for producing new DNA strands- Semiconservative, conservative, or dispersive replication?o Meselson-Stahl experiment: Grow E. coli cells in 15N nutrients, then transfer to 14N medium and allow to divide twice, extracting DNA each divisiono15N is heavier than 14N  perform density-gradient centrifugation and examineo Results: 1 band after first replication, 2 bands after 2 reps = semiconservative15.3 A model for DNA synthesis- DNA polymerase III polymerizes deoxyribonucleotides into DNA from 5’ to 3’- DNA synthesis is exergonic b/c monomers are deoxyribonuceloside triphosphates (dNTPs) which have high potential energy which is released as two phosphates are broken off- Replication bubble forms at the origin of replication (only one in bacteria, several in euks)o DNA synthesis is bidirectional, happening in two directions simultaneously- DNA helicase breaks bonds between base pairs to separate DNA strands- Single-strand DNA-binding proteins (SSBPs) prevent strands from rejoining- Topoisomerase cuts DNA, allows to unwind, and rejoins to relieve twisting tension- Primase (type of RNA polymerase) synthesizes a short primer for DNA polymeraseo Primers (RNA) provide 3’ hydroxyl group to join incoming nucleotides ontoo RNA polymerase catalyzes the polymerization of ribonucleotides into RNA- Sliding clamp holds DNA poly in place on strand- Leading/continuous strand leads into replication fork, synthesized continuously- Lagging/discontinuous strand lags behind synthesis at the forko Okazaki hypothesizes that primase makes new primers as the fork opens, and DNA polymerase makes short lagging-strand DNA that gets linked together Used pulse-chase strategy with deoxyribonucleotides to confirm existence of Okazaki fragments o Fragments connected by DNA polymerase I, which removes and replaces RNA primer DNA ligase then catalyzes formation of the phosphodiester bond- Replisome – a large macromolecular machine consisting of all the enzymes involved in DNA synthesis15.4 Replicating the ends of linear chromosomes- Telomere – the end of a eukaryotic chromosomeo Problem with lagging strands at the end of DNA, no DNA synthesis occurs after the RNA primer is removed and the resulting single-stranded DNA degrades (~50-100 bp)o Blackburn, Greider, and Szostak discover telomeres are made of repeated base sequences and that an enzyme called telomerase replicates telomeres using built-in RNA template Telomerase only active in some cell types, e.g. cells that produce gametes Somatic cells (cells not involved in gamete formation) lack telomerase activityo Greider tests somatic cells at various ages and discovers positive relationship between initial telomere length and number of cell divisions before cells stop dividingo Cancer cells have active telomerase, allowing unlimited divisions15.5 Repairing mistakes and DNA damage- Error rate during DNA replication about 1 per billion deoxyribonucleotides- DNA polymerase inserts incorrect deoxyribonucleotide once every ~100,000 basesadded- ε (epsilon) subunit of DNA polymerase III acts as an exonuclease (removes deoxyribonucleotides from ends of DNA strands)o Discovered by examining E. coli mutants with high error rates in replication –defect in DNA polymerase (specifically, in ε subunit)- Incorrect base pairing  wonky DNA geometry o DNA polymerase proofreads, only adds new nucleotide when previous shape lines upo If incorrect pair made, ε removes mismatched nucleotide and DNA poly proceeds- Mismatch repair occurs when mismatched bases are corrected after DNA synthesisfinisheso Discovered by analyzing E. coli mutants with abnormally high mutation rates mutS mutation causes a deficiency in mismatch repair (plays role in cancer)o Group of proteins recognize mismatched base, remove a section containing incorrect base, and fill in correct bases using untouched strand as template- Nucleotide excision repair system works on DNA damage caused by UV light and chemicalso UV light  covalent bond forms between adjacent pyrimidine bases on same strand Often creates “thymine dimers” which make a kink in DNA and block DNA replication by stalling DNA polymeraseso An enzyme recognizes the kink, another enzyme removes a segment of single-stranded DNA with the defect, DNA polymerase fills in the gap, ligase links together- Xeroderma pigmentosum (XP) is a rare autosomal recessive disease that causes extreme UV sensitivity in humanso J. Cleaver proposes people with XP have mutations in DNA nucleotide excision repair Used cell cultures to study XP cells, showed correlations that showed XP cells exposed to UV light could not repair damage- % cells survived vs. Dose of UV light- Amount of radioactive nucleotide incorporated vs. Dose of UV lighto If overall mutation rate in a cell is elevated b/c of defects in DNA repair, then mutations that trigger cancer become more likelyCHAPTER 16 NOTES: HOW GENES WORK- Gene expression – the process of converting archived information into molecules that do stuff16.1 What do genes do?- Beadle and Tatum’s research on Neurospora crassa to observe knock-out/null/loss-of-function alleles – alleles that don’t functiono Exposed N. crassa cells to radiation to damage DNA and located mutantso Led to one-gene, one-enzyme hypothesis – each gene contains the information needed to make an enzyme- Srb and Horowitz test one-gene, one-enzyme hypothesis by creating mutant cells and using a genetic screen – a technique for picking certain types of mutants out of manyo Raise colonies on arginine then transfer to medium without arginine, any cells


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Pitt BIOSC 0160 - Chapter 15

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