1 DNA and Replication

2 History of DNA

3 Early scientists thought protein was the cell’s hereditary material because it was more complex than DNA Proteins were composed of 20 different amino acids in long polypeptide chains

4 Genetic facts in 1900: Both female and male organisms have identical chromosomes except for one pair. Genes are located on chromosomes All organisms have two types of chromosomes: –Sex chromosomes –Autosomes

5 Male vs Female MALE Usually the Y chromosome. Y is usually smaller Male genotype = XY FEMALE Usually the X chromosome. Larger than the Y Female genotype XX Except Birds Male = XX Female = XY

6 Frederick Griffith British bacteriologist 1928 = designed and performed experiment on rats and bacteria that causes pneumonia. 2 strains of the bacteria Type S = causes severe pneumonia Type R = relatively harmless

7 Griffith’s Rats 1.First he injected living Type S bacteria into rats:

8 Second he injected dead Type S into the rats.

9 Next he injected living type R bacteria

10 Finally he injected a mixture of living Type R and dead Type S :

11 Results of experiments: Because the dead rat tissue showed living Type S bacteria, something “brought the Type S back to life” Actually one bacterial type incorporated the DNA, or instructions, from the dead bacteria into its own DNA Known as transformation. Confirmed by Avery, MacLeod, and McCarty in 1944

12 Oswald Avery Canadian biologist ( ) Discovered DNA in 1944 with a team of scientists.

13 Hershey and Chase 1952 Attempted to solve the debate on whether DNA or proteins are responsible for providing the genetic material.

14 They used a bacteriophage (a virus which attacks bacteria) to prove that DNA was definitely the genetic material.

15 Fig Bacterial cell Phage head Tail sheath Tail fiber DNA 100 nm

16 Fig EXPERIMENT Phage DNA Bacterial cell Radioactive protein Radioactive DNA Batch 1: radioactive sulfur ( 35 S) Batch 2: radioactive phosphorus ( 32 P) Empty protein shell Phage DNA Centrifuge Pellet Pellet (bacterial cells and contents) Radioactivity (phage protein) in liquid Radioactivity (phage DNA) in pellet

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18 History of DNA Chromosomes are made of both DNA and protein Experiments on bacteriophage viruses by Hershey & Chase proved that DNA was the cell’s genetic material Radioactive 32 P was injected into bacteria!

19 Phoebus A. Levene Russian born; immigrated to America, moves to Europe. 1920’s discovered nucleotides (building blocks of DNA) 1.Sugar 2.Phosphate group 3.Nitrogenous base

20 Composition of DNA

21 Components of DNA A very long molecule. 4 nitrogenous bases:

22 Chargaff’s rules The relative amounts of adenine and thymine are the same in DNA The relative amounts of cytosine and guanine are the same. Named after Erwin Chargaff

23 Chargaff’s Rule Adenine ThymineAdenine must pair with Thymine Guanine CytosineGuanine must pair with Cytosine The bases form weak hydrogen bonds G C TA

24 Discovery of DNA Structure Erwin Chargaff showed the amounts of the four bases on DNA ( A,T,C,G) In a body or somatic cell: A = 30.3% T = 30.3% G = 19.5% C = 19.9%

25 Question: Adenine CytosineIf there is 30% Adenine, how much Cytosine is present?

26 Answer: CytosineThere would be 20% Cytosine Adenine (30%) = Thymine (30%)Adenine (30%) = Thymine (30%) Guanine (20%) = Cytosine (20%)Guanine (20%) = Cytosine (20%) Therefore, 60% A-T and 40% C-GTherefore, 60% A-T and 40% C-G

27 DNA Structure Rosalind Franklin took diffraction x-ray photographs of DNA crystals In the 1950’s, Watson & Crick built the first model of DNA using Franklin’s x-rays

28 Rosalind Franklin

29 Rosalind Franklin Used X-Ray diffraction to get information about the structure of DNA:

30 Fig. 16-6a (a) Rosalind Franklin

31 Fig. 16-6b (b) Franklin’s X-ray diffraction photograph of DNA

32 Structure of DNA Discovered in 1953 by two scientists: James Watson (USA) Francis Crick (GBR) Known as the double-helix model.

33 Fig. 16-1

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35 The double-helix A twisted ladder with two long chains of alternating phosphates and sugars. The nitrogenous bases act as the “rungs” joining the two strands.

36 How long is the DNA molecule?

37 Chromosomes & DNA replication The nucleus of one human cell contains approximately 1 meter of DNA. Histones = DNA tightly wrapped around a protein Nucleosome:

38 Chromosome structure:

39 DNA Structure

40 DNA Two strands coiled called a double helix Sides made of a pentose sugar Deoxyribose bonded to phosphate (PO 4 ) groups by phosphodiester bonds Center made of nitrogen bases bonded together by weak hydrogen bonds

41 DNA Double Helix Nitrogenous Base (A,T,G or C) “Rungs of ladder” “Legs of ladder” Phosphate & Sugar Backbone

42 Helix Most DNA has a right-hand twist with 10 base pairs in a complete turnMost DNA has a right-hand twist with 10 base pairs in a complete turn Left twisted DNA is called Z-DNA or southpaw DNALeft twisted DNA is called Z-DNA or southpaw DNA Hot spots occur where right and left twisted DNA meet producing mutationsHot spots occur where right and left twisted DNA meet producing mutations

43 DNA Stands for Deoxyribonucleic acid nucleotidesMade up of subunits called nucleotides Nucleotide made of:Nucleotide made of: Phosphate group 1.Phosphate group 5-carbon sugar 2.5-carbon sugar Nitrogenous base 3.Nitrogenous base

44 DNA Nucleotide O=P-O OPhosphate Group Group N Nitrogenous base (A, G, C, or T) (A, G, C, or T) CH2 O C1C1 C4C4 C3C3 C2C2 5 Sugar Sugar(deoxyribose) O

45 Pentose Sugar Carbons are numbered clockwise 1’ to 5’ CH2 O C1C1 C4C4 C3C3 C2C2 5 Sugar Sugar(deoxyribose)

46 DNA P P P O O O P P P O O O G C TA

47 Antiparallel Strands One strand of DNA goes from 5’ to 3’ (sugars) The other strand is opposite in direction going 3’ to 5’ (sugars)

48 Nitrogenous Bases Double ring PURINESDouble ring PURINES Adenine (A) Guanine (G) Single ring PYRIMIDINESSingle ring PYRIMIDINES Thymine (T) Cytosine (C) T or C A or G

49 Base-Pairings Purines only pair with Pyrimidines Three hydrogen bonds required to bond Guanine & Cytosine CG 3 H-bonds

50 T A Two hydrogen bonds are required to bond Adenine & Thymine

51 Fig. 16-7a Hydrogen bond 3 end 5 end 3.4 nm 0.34 nm 3 end 5 end (b) Partial chemical structure(a) Key features of DNA structure 1 nm

52 Fig. 16-UN1 Purine + purine: too wide Pyrimidine + pyrimidine: too narrow Purine + pyrimidine: width consistent with X-ray data

53 The Basic Principle: Base Pairing to a Template Strand Since the two strands of DNA are complementary, each strand acts as a template for building a new strand in replication In DNA replication, the parent molecule unwinds, and two new daughter strands are built based on base-pairing rules Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Animation: DNA Replication Overview Animation: DNA Replication Overview

54 DNA Replication

55 Fig A T G C TA TA G C (a) Parent molecule

56 Fig A T G C TA TA G C A T G C T A T A G C (a) Parent molecule (b) Separation of strands

57 Fig A T G C TA TA G C (a) Parent molecule AT GC T A T A GC (c) “Daughter” DNA molecules, each consisting of one parental strand and one new strand (b) Separation of strands A T G C TA TA G C A T G C T A T A G C

58 Replication Facts DNA has to be copied before a cell dividesDNA has to be copied before a cell divides New cells will need identical DNA strandsNew cells will need identical DNA strands

59 DNA replication Why needed? Step 1: DNA unzips. DNA polymerase helps in unzipping Starts at many different points.

60 DNA Replication Step 2:Step 2: Begins at Origins of ReplicationBegins at Origins of Replication Two strands open forming Replication Forks (Y-shaped region)Two strands open forming Replication Forks (Y-shaped region) New strands grow at the forksNew strands grow at the forks ReplicationFork Parental DNA Molecule 3’ 5’ 3’ 5’

61 DNA Replication Step 3:Step 3: As the 2 DNA strands open at the origin, Replication Bubbles formAs the 2 DNA strands open at the origin, Replication Bubbles form Human chromosomes have MANY bubbles but bacteria have one. Bubbles

62 DNA Replication Step 4:Step 4: Enzyme Helicase unwinds and separates the 2 DNA strandsEnzyme Helicase unwinds and separates the 2 DNA strands Single-Strand Binding ProteinsSingle-Strand Binding Proteins keep the 2 DNA strands separated and untwisted

63 DNA Replication Step 5: RNA primersStep 5: RNA primers start the addition of new nucleotides Step 6: DNA polymerase can then add and finish adding the new nucleotides

64 Fig Topoisomerase Helicase Primase Single-strand binding proteins RNA primer

65 DNA Replication DNA polymerase can only add nucleotides to the 3’ end of the DNADNA polymerase can only add nucleotides to the 3’ end of the DNA This causes the NEW strand to be built in a 5’ to 3’ directionThis causes the NEW strand to be built in a 5’ to 3’ direction RNAPrimer DNA Polymerase Nucleotide 5’ 3’ Direction of Replication

66 Remember the Strands are Antiparallel P P P O O O P P P O O O G C TA

67 Synthesis of the New DNA Strands Step 7: The Leading StrandStep 7: The Leading Strand is added in 5 to 3 direction RNAPrimer DNA Polymerase Nucleotides 3’5’

68 Step 8: The Lagging Strand is discontinuouslyStep 8: The Lagging Strand is added discontinuously on 3 to 5 direction. Step 9: This strand has many short segments called OKAZAKI fragments RNA Primer Leading Strand DNA Polymerase 5’5’ 5’ 3’ Lagging Strand 5’ 3’

69 Joining of Okazaki Fragments Step 10: The enzyme Ligase joins the Okazaki fragments together to make one strandStep 10: The enzyme Ligase joins the Okazaki fragments together to make one strand Lagging Strand Okazaki Fragment 2 DNA ligase DNA ligase Okazaki Fragment 1 5’ 3’

70 Fig Overview Origin of replication Leading strand Lagging strand Overall directions of replication Leading strand Lagging strand Helicase Parental DNA DNA pol III PrimerPrimase DNA ligase DNA pol III DNA pol I Single-strand binding protein

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72 Completing the replication After the DNA molecule comes apart, bases of free nucleotides in the nucleus join their complimentary bases.

73 Proofreading New DNA DNA polymerase initially makes about 1 in 10,000 base pairing errorsDNA polymerase initially makes about 1 in 10,000 base pairing errors Enzymes proofread and correct these mistakesEnzymes proofread and correct these mistakes The new error rate for DNA that has been proofread is 1 in 1 billion base pairing errorsThe new error rate for DNA that has been proofread is 1 in 1 billion base pairing errors

74 Semiconservative Model of Replication Idea presented by Watson & CrickIdea presented by Watson & Crick TheThe two strands of the parental molecule separate, and each acts as a template for a new complementary strand New DNA consists of 1 PARENTAL (original) and 1 NEW strand of DNA Parental DNA DNA Template New DNA

75 DNA Damage & Repair Chemicals & ultraviolet radiation damage the DNA in our body cells Cells must continuously repair DAMAGED DNA Excision repair occurs when any of over 50 repair enzymes remove damaged parts of DNA DNA polymerase and DNA ligase replace and bond the new nucleotides together

76 Question: What would be the complementary DNA strand for the following DNA sequence? DNA 5’-CGTATG-3’

77 Answer: DNA 5’-GCGTATG-3’ DNA 3’-CGCATAC-5’

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