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Transcript
DNA and Replication 1 History of DNA 2 History of DNA • 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 3 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 4 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 5 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 6 Griffith’s Rats 1. First he injected living Type S bacteria into rats: 7 • Second he injected dead Type S into the rats. 8 • Next he injected living type R bacteria 9 • Finally he injected a mixture of living Type R and dead Type S : 10 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 11 Oswald Avery • Canadian biologist (18771955) • Discovered DNA in 1944 with a team of scientists. 12 Hershey and Chase • 1952 • Attempted to solve the debate on whether DNA or proteins are responsible for providing the genetic 13 • They used a bacteriophage (a virus which attacks bacteria) to prove that DNA was definitely the genetic material. 14 Fig. 16-3 Phage head Tail sheath Tail fiber 100 nm DNA Bacterial cell 15 Fig. 16-4-3 EXPERIMENT Phage Empty protein Radioactive shell protein Radioactivity (phage protein) in liquid Bacterial cell Batch 1: radioactive sulfur (35S) DNA Phage DNA Centrifuge Pellet (bacterial cells and contents) Radioactive DNA Batch 2: radioactive phosphorus (32P) Centrifuge Pellet Radioactivity (phage DNA) in pellet 16 17 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 32P was injected into bacteria! 18 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 19 Composition of DNA 20 Components of DNA • A very long molecule. 4 nitrogenous bases: 21 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 22 Chargaff’s Rule • Adenine must pair with Thymine • Guanine must pair with Cytosine • The bases form weak hydrogen bonds T A G C 23 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% 24 Question: • If there is 30% Adenine, how much Cytosine is present? 25 Answer: • There would be 20% Cytosine • Adenine (30%) = Thymine (30%) • Guanine (20%) = Cytosine (20%) • Therefore, 60% A-T and 40% C-G 26 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 27 Rosalind Franklin 28 Rosalind Franklin • Used X-Ray diffraction to get information about the structure of DNA: 29 Fig. 16-6a (a) Rosalind Franklin 30 Fig. 16-6b (b) Franklin’s X-ray diffraction photograph of DNA 31 Structure of DNA • Discovered in 1953 by two scientists: • James Watson (USA) • Francis Crick (GBR) • Known as the double-helix model. 32 Fig. 16-1 33 34 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. 35 How long is the DNA molecule? 36 Chromosomes & DNA replication • The nucleus of one human cell contains approximately 1 meter of DNA. • Histones = DNA tightly wrapped around a protein • Nucleosome: 37 Chromosome structure: 38 DNA Structure 39 DNA • Two strands coiled called a double helix • Sides made of a pentose sugar Deoxyribose bonded to phosphate (PO4) groups by phosphodiester bonds • Center made of nitrogen bases bonded together by weak hydrogen bonds 40 DNA Double Helix “Rungs of ladder” Nitrogenous Base (A,T,G or C) “Legs of ladder” Phosphate & Sugar Backbone 41 Helix • Most DNA has a right-hand twist with 10 base pairs in a complete turn • Left twisted DNA is called Z-DNA or southpaw DNA • Hot spots occur where right and left twisted DNA meet producing mutations 42 DNA • Stands for Deoxyribonucleic acid • Made up of subunits called nucleotides • Nucleotide made of: 1. Phosphate group 2. 5-carbon sugar 3. Nitrogenous base 43 DNA Nucleotide Phosphate Group O O=P-O O 5 CH2 O N C1 C4 Sugar (deoxyribose) C3 C2 Nitrogenous base (A, G, C, or T) 44 Pentose Sugar • Carbons are numbered clockwise 1’ to 5’ 5 CH2 O C1 C4 Sugar (deoxyribose) C3 C2 45 5 DNA O 3 3 P 5 O O C G 1 P 5 3 2 4 4 P 5 P 2 3 1 O T A 3 O 3 5 O 5 P P 46 Antiparallel Strands • One strand of DNA goes from 5’ to 3’ (sugars) • The other strand is opposite in direction going 3’ to 5’ (sugars) 47 Nitrogenous Bases • Double ring PURINES Adenine (A) Guanine (G) A or G • Single ring PYRIMIDINES Thymine (T) Cytosine (C) T or C 48 Base-Pairings • Purines only pair with Pyrimidines • Three hydrogen bonds required to bond Guanine & Cytosine 3 H-bonds G C 49 •Two hydrogen bonds are required to bond Adenine & Thymine T A 50 Fig. 16-7a 5 end Hydrogen bond 3 end 1 nm 3.4 nm 3 end 0.34 nm (a) Key features of DNA structure (b) Partial chemical structure 5 end 51 Fig. 16-UN1 Purine + purine: too wide Pyrimidine + pyrimidine: too narrow Purine + pyrimidine: width consistent with X-ray data 52 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 Animation: DNA Replication Overview Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 53 DNA Replication 54 Fig. 16-9-1 A T C G T A A T G C (a) Parent molecule 55 Fig. 16-9-2 A T A T C G C G T A T A A T A T G C G C (a) Parent molecule (b) Separation of strands 56 Fig. 16-9-3 A T A T A T A T C G C G C G C G T A T A T A T A A T A T A T A T G C G C G C G C (a) Parent molecule (b) Separation of strands (c) “Daughter” DNA molecules, each consisting of one parental strand and one new strand 57 Replication Facts • DNA has to be copied before a cell divides • New cells will need identical DNA strands 58 DNA replication Why needed? • Step 1: DNA unzips. • DNA polymerase helps in unzipping • Starts at many different points. 59 DNA Replication • Step 2: • Begins at Origins of Replication • Two strands open forming Replication Forks (Y-shaped region) 3’ • New strands grow at the forks 5’ Parental DNA Molecule 3’ Replication Fork 60 5’ DNA Replication • Step 3: • As the 2 DNA strands open at the origin, Replication Bubbles form • Human chromosomes have MANY bubbles but bacteria have one. Bubbles Bubbles 61 DNA Replication • Step 4: • Enzyme Helicase unwinds and separates the 2 DNA strands • Single-Strand Binding Proteins keep the 2 DNA strands separated and untwisted 62 DNA Replication • Step 5: RNA primers start the addition of new nucleotides • Step 6: DNA polymerase can then add and finish adding the new nucleotides 63 Fig. 16-13 Primase Single-strand binding proteins 3 Topoisomerase 5 RNA primer 5 3 5 Helicase 64 3 DNA Replication • DNA 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’ direction 5’ 3’ Nucleotide DNA Polymerase Direction of Replication RNA Primer 65 5’ Remember the Strands are Antiparallel 5 O 3 3 P 5 O O C G 1 P 5 3 2 4 4 P 5 P 2 3 1 O T A 3 O 3 5 O 5 P P 66 Synthesis of the New DNA Strands • Step 7: The Leading Strand is added in 5 to 3 direction 5’ 3’ Nucleotides DNA Polymerase 5’ RNA Primer 67 • Step 8: The Lagging Strand is added discontinuously on 3 to 5 direction. • Step 9: This strand has many short segments called OKAZAKI fragments Leading Strand 5 ’ 3’ DNA Polymerase 5’ 3’ Lagging Strand RNA Primer 3’ 5’ 3’ 5’ 68 Joining of Okazaki Fragments • Step 10: The enzyme Ligase joins the Okazaki fragments together to make one strand DNA ligase 5’ 3’ Okazaki Fragment 1 Okazaki Fragment 2 3’ 5’ Lagging Strand 69 Fig. 16-17 Overview Origin of replication Lagging strand Leading strand Leading strand Lagging strand Overall directions of replication Single-strand binding protein Helicase 5 Leading strand 3 DNA pol III 3 Parental DNA Primer 5 Primase 3 DNA pol III Lagging strand 5 4 DNA pol I 3 5 3 DNA ligase 2 1 70 3 5 71 Completing the replication • After the DNA molecule comes apart, bases of free nucleotides in the nucleus join their complimentary bases. 72 Proofreading New DNA • DNA polymerase initially makes about 1 in 10,000 base pairing errors • Enzymes proofread and correct these mistakes • The new error rate for DNA that has been proofread is 1 in 1 billion base pairing errors 73 Semiconservative Model of Replication • Idea presented by Watson & Crick • The 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 DNA Template strand of DNA Parental DNA New DNA 74 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 75 Question: • What would be the complementary DNA strand for the following DNA sequence? DNA 5’-CGTATG-3’ 76 Answer: DNA 5’-GCGTATG-3’ DNA 3’-CGCATAC-5’ 77 78