Brief History. Many people contributed to our understanding of DNA

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1 DNA (Ch. 16)

2 Brief History Many people contributed to our understanding of DNA T.H. Morgan (1908) Frederick Griffith (1928) Avery, McCarty & MacLeod (1944) Erwin Chargaff (1947) Hershey & Chase (1952) Watson & Crick (1953) Meselson & Stahl (1958)

3 Chromosomes related to phenotype T.H. Morgan working with Drosophila associated phenotype with specific chromosome white-eyed male had specific X chromosome

4 Avery, McCarty & MacLeod Conclusion first experimental evidence that DNA was the genetic material Oswald Avery Maclyn McCarty Colin MacLeod

5 Martha Chase Alfred Hershey Hershey & Chase Hershey

6 Erwin Chargaff 1947 DNA composition: Chargaff s rules varies from species to species all 4 bases not in equal quantity bases present in characteristic ratio humans: A = 30.9% T = 29.4% G = 19.9% C = 19.8% That s interesting! What do you notice? Rules A = T C = G

7 Consider This Brief Film Strip

8 Structure of DNA Watson & Crick developed double helix model of DNA other leading scientists working on question: Rosalind Franklin Maurice Wilkins Linus Pauling Franklin Wilkins Pauling

9 Watson and Crick 1953 article in Nature Watson Crick

10 Rosalind Franklin ( )

11 It s Simple, No?

12 But how is DNA copied? Replication of DNA base pairing suggests that it will allow each side to serve as a template for a new strand It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material. Watson & Crick

13 Meselson & Stahl Matthew Meselson Franklin Stahl Franklin Stahl Matthew Meselson

14 Scientific History March to understanding that DNA is the genetic material T.H. Morgan (1908): genes are on chromosomes Frederick Griffith (1928): a transforming factor can change phenotype Avery, McCarty & MacLeod (1944): transforming factor is DNA Erwin Chargaff (1947): Chargaff rules: A = T, C = G Hershey & Chase (1952): confirmation that DNA is genetic material Watson & Crick (1953): determined double helix structure of DNA Meselson & Stahl (1958): semi-conservative replication

15 The Central Dogma Flow of genetic information in a cell transcription translation DNA RNA protein replication

16 Directionality of DNA You need to number the carbons! it matters! PO 4 nucleotide N base This will be IMPORTANT!! 4 CH 2 O ribose 1 OH 2

17 Watson and Crick 1953 article in Nature

18 Double helix structure of DNA It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material. Watson & Crick

19 The DNA backbone Putting the DNA backbone together refer to the and ends of the DNA the last trailing carbon Sounds trivial, but this will be IMPORTANT!! PO 4 CH 2 4 O C O O P O O CH 2 O 4 2 base 1 base 1 OH 2

20 Anti-parallel strands Nucleotides in DNA backbone are bonded from phosphate to sugar between & carbons DNA molecule has direction complementary strand runs in opposite direction

21 Bonding in DNA hydrogen bonds covalent phosphodiester bonds.strong or weak bonds? How do the bonds fit the mechanism for copying DNA?

22 Base pairing in DNA Purines adenine (A) guanine (G) Pyrimidines thymine (T) cytosine (C) Pairing A : T 2 bonds C : G 3 bonds

23 Copying DNA Replication of DNA base pairing allows each strand to serve as a template for a new strand new strand is 1/2 parent template & 1/2 new DNA semi-conservative copy process

24 DNA Replication Let s meet the team Large team of enzymes coordinates replication

25 Replication: 1st step Unwind DNA helicase enzyme unwinds part of DNA helix stabilized by single-stranded binding proteins helicase single-stranded binding proteins replication fork

26 Replication: 2nd step Build daughter DNA strand add new complementary bases DNA polymerase III DNA Polymerase III

27 Energy of Replication Where does energy for bonding usually come from? You remember ATP! Are there other energy ways nucleotides? to get energy You out of bet! it? We come with our own energy! energy energy GTP TTP CTP ATP modified nucleotide And we leave behind a nucleotide! ADP AMP GMP TMP CMP

28 Replication Adding bases can only add nucleotides to end of a growing DNA strand need a starter nucleotide to bond to strand only grows energy DNA Polymerase III energy DNA Polymerase III energy DNA Polymerase III DNA Polymerase III energy

29 Leading & Lagging strands Okazaki Limits of DNA polymerase III can only build onto end of an existing DNA strand ligase Lagging strand growing replication fork Lagging strand Okazaki fragments joined by ligase spot welder enzyme DNA polymerase III Leading strand Leading strand continuous synthesis

30 Replication fork / Replication bubble DNA polymerase III leading strand lagging strand growing replication fork lagging strand leading strand leading strand lagging strand growing replication fork

31 Replacing RNA primers with DNA DNA polymerase I removes sections of RNA primer and replaces with DNA nucleotides DNA polymerase I ligase growing replication fork RNA But DNA polymerase I still can only build onto end of an existing DNA strand

32 Chromosome erosion Houston, we have a problem! All DNA polymerases can only add to end of an existing DNA strand DNA polymerase I growing replication fork DNA polymerase III RNA Loss of bases at ends in every replication chromosomes get shorter with each replication limit to number of cell divisions?

33 Telomeres Repeating, non-coding sequences at the end of chromosomes = protective cap limit to ~50 cell divisions growing replication fork telomerase Telomerase enzyme extends telomeres can add DNA bases at end different level of activity in different cells high in stem cells & cancers -- Why? TTAAGGG TTAAGGG

34 Replication fork DNA polymerase I DNA polymerase III Okazaki fragments lagging strand 5 ligase 3 5 primase 3 SSB 3 helicase leading strand direction of replication DNA polymerase III SSB = single-stranded binding proteins

35 DNA polymerases DNA polymerase III 1000 bases/second! main DNA builder DNA polymerase I 20 bases/second editing, repair & primer removal DNA polymerase III enzyme Thomas Kornberg?? Arthur Kornberg 1959

36 Editing & proofreading DNA 1000 bases/second = lots of typos! DNA polymerase I proofreads & corrects typos repairs mismatched bases removes abnormal bases repairs damage throughout life reduces error rate from 1 in 10,000 to 1 in 100 million bases

37 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 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

38 What does it really look like?

39 What Does It Really Look Like, (3D Animated Movie Version)

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