Chapter 16. DNA The Genetic Material Replication
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1 Chapter 16. DNA The Genetic Material Replication Scientific History The march to understanding that DNA is the genetic material T.H. Morgan (1908) Frederick Griffith (1928) Avery, McCarty & MacLeod (1944) Hershey & Chase (1952) Watson & Crick (1953) Meselson & Stahl (1958) 1
2 Genes are on chromosomes T.H. Morgan working with Drosophila (fruit flies) genes are on chromosomes but is it the protein or the DNA of the chromosomes that are the genes? through 1940 proteins were thought to be genetic material Why? The Transforming Factor Frederick Griffith Streptococcus pneumonia bacteria was working to find cure for pneumonia harmless live bacteria mixed with heatkilled infectious bacteria causes disease in mice substance passed from dead bacteria to live bacteria = Transforming Factor
3 The Transforming Factor Transformation: change in genotype & phenotype due to assimilation of external DNA by a cell 1944 DNA is the Transforming Factor Avery, McCarty & MacLeod purified both DNA & proteins from Streptococcus pneumonia bacteria which will transform non-pathogenic bacteria? injected protein into bacteria no effect injected DNA into bacteria transformed harmless bacteria into virulent bacteria What s the conclusion? 3
4 Avery, McCarty & MacLeod Oswald Avery Colin MacLeod Maclyn McCarty Confirmation of DNA Hershey & Chase classic blender experiment worked with bacteriophage viruses that infect bacteria grew phage viruses in 2 media, radioactively labeled with either: 35 S in their proteins 32 P in their DNA infected bacteria with labeled phages
5 Hershey & Chase Martha Chase Alfred Hershey 5
6 Blender experiment Radioactive phage & bacteria in blender 35 S phage radioactive proteins stayed in supernatant therefore protein did NOT enter bacteria 32 P phage radioactive DNA stayed in pellet therefore DNA did enter bacteria DNA is confirmed as transforming factor 6
7 1947 Chargaff DNA composition: Chargaff s rules varies from species to species amounts of 4 bases not equal bases present in characteristic ratio humans: A=30.9% T=29.4% G=19.9% C=19.8% Structure of DNA Watson & Crick developed double helix model of DNA other scientists working on question: Linus Pauling Maurice Wilkins Rosalind Franklin
8 Rosalind Franklin ( ) Watson and Crick 1953 article in Nature 8
9 Double helix structure of DNA the structure of DNA suggested a mechanism for how DNA is copied by the cell Anti-parallel strands Phosphate to sugar bond involves carbons in 3 & 5 positions DNA molecule has direction complementary strand runs in opposite direction It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material. 9
10 Bonding in DNA hydrogen 5 bonds 3 phosphodiester bonds 3 5.strong or weak bond? How do the bonds fit the mechanism for copying DNA? Base pairing in DNA Purines adenine guanine Pyrimidines thymine cytosine Pairing A : T C : G 10
11 Copying DNA Replication of DNA base pairing allows each strand to serve as a pattern for a new strand Models of DNA Replication Alternative models needed to be verified through experimentation 11
12 1958 Semi-conservative replication Meselson & Stahl labeled nucleotides of parent DNA strands with heavy nitrogen = 15 N labeled new nucleotides with lighter isotope = 14 N replicated strands were found to be half 15 N & half 14 N The Most Beautiful Experiment in Biology DNA Replication Large team of enzymes coordinates replication 12
13 Helicase Opens DNA helix enabling replication DNA Polymerase III Adds nucleotides only to 3 end nucleoside-p-p-p links to sugar-p backbone losing 2-P provides energy for bonding Does this sound familiar?? 13
14 A common thread in biology Why 3Ps? 5 3 You don t just get energy from ATP ADP TTP TMP GTP GMP CTP CMP 3 5 Leading & Lagging strands Leading strand continuous synthesis Lagging strand Okazaki fragments joined by ligase spot welder 14
15 Okazaki fragments Priming DNA synthesis DNA polymerase can only extend an existing DNA molecule. Cannot start a new one. short RNA primer is built first on parent DNA strand by primase RNA primer later removed by DNA polymerase I 15
16 Replication fork DNA polymerase lagging strand ligase Okazaki primase fragments SSB 5 3 leading strand direction of replication 3 DNA polymerase 3 helicase 5 Replication bubble Adds 1000 bases/second! Which direction does DNA build? List the enzymes & their role 16
17 Replication enzymes DNA polymerases DNA polymerase I 20 bases/second editing, repair & primer removal DNA polymerase III 1000 bases/second main DNA building enzyme 17
18 Editing & proofreading DNA At 1000 bases/second, lots of typos! DNA polymerase I excises mismatched bases reduces error rate from 1 in 10,000 to 1 in 100 million bases Fast & accurate! It takes E. coli <1 hour to copy 5 million base pairs in its single chromosome & divide to form 2 identical daughter cells Human cell copies its 6 billion bases & divide into daughter cells in only few hours remarkably accurate only ~1 error per 100 million bases ~30 errors per cell cycle 18
19 Problems in the end Ends of chromosomes are eroded with each replication Telomeres expendable, non-coding sequences at ends of DNA short sequence of bases repeated 1000s times TTAGGG in humans Telomerase enzyme in certain cells 19
20 The Central Dogma flow of genetic information within a cell DNA transcription RNA translation protein replication 20
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