The Molecular Basis of Inheritance

Transcription

1 The Molecular Basis of Inheritance

2 Scientific History The march to understanding that DNA is the genetic material T.H. Morgan (1908) Frederick Griffith (1928) Avery, McCarty & MacLeod (1944) Erwin Chargaff (1947) Hershey & Chase (1952) Watson & Crick (1953) Meselson & Stahl (1958)

3 The Transforming Principle 1928 Frederick Griffith Streptococcus pneumonia bacteria was working to find cure for pneumonia harmless live bacteria ( rough ) mixed with heat-killed pathogenic bacteria ( smooth ) causes fatal disease in mice a substance passed from dead bacteria to live bacteria to change their phenotype Transforming Principle

4 Transformation = change in phenotype something in heat-killed bacteria could still transmit disease-causing properties The Transforming Principle mix heat-killed pathogenic & live pathogenic live non-pathogenic heat-killed non-pathogenic strain of bacteria strain of bacteria pathogenic bacteria bacteria A. B. C. D. mice die mice live mice live mice die

5 DNA is the Transforming Principle Avery, McCarty & MacLeod purified both DNA & proteins separately 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 1944 mice die

6 Avery, McCarty & MacLeod 1944??!! Conclusion first experimental evidence that DNA was the genetic material Oswald Avery Maclyn McCarty Colin MacLeod

7 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 Hershey

8 Hershey & Protein coat labeled with 35 S T2 bacteriophages are labeled with radioactive isotopes S vs. P DNA labeled with 32 P Chase bacteriophages infect bacterial cells Which radioactive marker is found inside the cell? bacterial cells are agitated to remove viral protein coats Which molecule carries viral genetic info? 35 S radioactivity found in the medium 32 P radioactivity found in the bacterial cells

9

10 Blender experiment Radioactive phage & bacteria in blender 35 S phage radioactive proteins stayed in supernatant therefore viral protein did NOT enter bacteria 32 P phage radioactive DNA stayed in pellet therefore viral DNA did enter bacteria Confirmed DNA is transforming factor

11 Martha Chase Alfred Hershey Hershey & Chase Hershey

12 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%

13 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

14 Watson and Crick 1953 article in Nature Watson Crick

15 Rosalind Franklin ( )

16 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

17 Directionality of DNA You need to number the carbons! it matters! PO 4 nucleotide N base CH 2 O 4ʹ ribose 1ʹ OH 2ʹ

18 The DNA backbone Putting the DNA backbone together refer to the and ends of the DNA the last trailing carbon PO 4 CH 2 4ʹ O C O O P O O CH 2 O 4ʹ 2ʹ base 1ʹ base 1ʹ OH 2ʹ

19 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

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

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

22 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

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 Semiconservative replication, when a double helix replicates each of the daughter molecules will have one old strand and one newly made strand. Experiments in the late 1950s by Matthew Meselson and Franklin Stahl supported the semiconservative model, proposed by Watson and Crick, over the other two models. (Conservative & dispersive)

25 DNA Replication Large team of enzymes coordinates replication

26

27 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

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

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

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

31 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

32 Starting DNA synthesis: RNA Limits of DNA polymerase III u can only build onto end of an existing DNA strand primers growing replication fork DNA polymerase III primase RNA RNA primer u u built by primase serves as starter sequence for DNA polymerase III

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

34 Chromosome erosion 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 u u chromosomes get shorter with each replication limit to number of cell divisions?

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

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

37 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 Roger Kornberg 2006 Arthur Kornberg 1959

38 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

39 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

40 What does it really look like?

41