BIOLOGY 2 CHAPTER 15 NOTES DNA AND THE GENE SYNTHESIS AND REPAIR 15 1 What are genes made of structure function DNA or protein Originally thought proteins b c limitless variation in o Avery showed early on that DNA could be genetic material o 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 pairing 15 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 division o 15N is heavier than 14N perform density gradient centrifugation and examine o Results 1 band after first replication 2 bands after 2 reps semiconservative 15 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 polymerase o Primers RNA provide 3 hydroxyl group to join incoming nucleotides onto o 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 fork o 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 synthesis 15 4 Replicating the ends of linear chromosomes Telomere the end of a eukaryotic chromosome o 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 Somatic cells cells not involved in gamete formation lack telomerase gametes activity o Greider tests somatic cells at various ages and discovers positive relationship between initial telomere length and number of cell divisions before cells stop dividing o Cancer cells have active telomerase allowing unlimited divisions 15 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 bases added 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 subunit Incorrect base pairing wonky DNA geometry o DNA polymerase proofreads only adds new nucleotide when previous shape o If incorrect pair made removes mismatched nucleotide and DNA poly Mismatch repair occurs when mismatched bases are corrected after DNA synthesis o 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 o UV light covalent bond forms between adjacent pyrimidine bases on same lines up proceeds finishes chemicals strand Often creates thymine dimers which make a kink in DNA and block DNA replication by stalling DNA polymerases o 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 humans o 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 light o If overall mutation rate in a cell is elevated b c of defects in DNA repair then mutations that trigger cancer become more likely CHAPTER 16 NOTES HOW GENES WORK Gene expression the process of converting archived information into molecules that do stuff 16 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 function o Exposed N crassa cells to radiation to damage DNA and located mutants o 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 many o Raise colonies on arginine then transfer to medium without arginine any cells that can grow in medium w arginine but not medium w o arginine cannot make its own arginine Repeat with ornithine and citrulline o Confirmed one gene one enzyme hypothesis and that genes are
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