DNA - Deoxyribonucleic Awesomeness! PDF

DNA DeoxyRibonucleic Awesomeness! 1 Part 1. Discovering the Heritable Material: Experimental Approaches 2  Discovering the Heritable Material: Experimental Approaches What is the genetic mate...

DNA DeoxyRibonucleic Awesomeness! 1 Part 1. Discovering the Heritable Material: Experimental Approaches 2  Discovering the Heritable Material: Experimental Approaches What is the genetic material? – DNA? Protein? Independent Learning Ch 11: Three key experiments in determining DNA was the genetic material: – Griffith experiment 1928 – Avery, McCarty and Macleod 1944 – Hershey and Chase 1952 3 3  Griffith experiment Documented movement of heritable material transmitted from one organism to another (he called this, transformation and was convinced that it involved movement of a protein – 2 strains of S. pneumonia  smooth (lethal) S-strain  rough (nonlethal) R-strain – Injected the strains into mice 4 4  Live Dead Dead Smooth Live Rough Smooth Smooth & Live Rough Bacteria Bacteria Bacteria Bacteria What happens to mice? ? ? Smooth cells cultured Smooth cells cultured from the body from the body Smooth: had genes for a polysaccharide capsule which inhibited the mouse’s immune system Rough: lacked one of the genes and therefore had no capsule, so less virulent 5  Live Dead Dead Smooth Live Rough Smooth Smooth & Live Rough Bacteria Bacteria Bacteria Bacteria Griffith showed that live R bacteria could be converted to virulent S bacteria: molecules from dead S cells could genetically transform the R bacteria to take on Smooth capsule property - called these molecules the “transforming principle” (so transf. principle was the genetic material, but still did not know what it was composed of…) 6  Avery, McCarty, and MacLeod Extended Griffith’s work – what is chemical nature of transforming principle? Added different macromolecules from an extract of heat-killed S strain to R-strain to see which macromolecule was necessary for transformation – Polysaccharide coat hydrolyzed R to S transformation – Proteins hydrolyzed  R to S transformation still occurs – Nucleic acids hydrolyzed  NO transformation *Trans.Principle identified, Must be nucleic acid!* Which nucleic acid?  RNA was unable to transform R strain to S strain  DNA could transform R strain to S strain  *Therefore Transforming.Principle =DNA 7 7  Hershey and Chase Experiments They labeled DNA and protein (separately) with radioactive isotope tracer /tag: – 35 S labeled proteins (no S in DNA) – 32 P labeled only DNA (no P in protein) 2 protocols: infected 2 colonies of E.coli, each with a different tag (35S or 32P) – upon infection, viral genetic material (heritable) enters host bacterial cell – disruption in blender removes non-transferred material – They tested progeny phages for radioactivity 32 P colony: radioactivity entered cells 35 S colony: radioactivity stayed outside the cell 8 (protein coat does not enter) 8  35 S label (protein) No 35S label in progeny phages label does not enter cell 32 P label (DNA) 32 P Label in progeny phages label enters cell DNA must contain the genetic instructions for new virus formation Confirmed DNA is genetic / heritable material of living organisms http://highered.mcgraw-hill.com/sites/9834092 ment.html 9  At Home Exercise The following three research efforts helped discover that DNA was the genetic material. Write brief notes on what each of the following experiments involved and the main results/conclusion: – Griffith experiment – Avery, McCarty and Macleod experiment – Hershey and Chase experiment 10 In the Griffith experiment, which combination(s) would result in a dead mouse? A) Live Rough bacteria injected B) Live Smooth bacteria injected C) Heat-killed Smooth bacteria injected D) Heat-killed Smooth bacteria plus Live Rough bacteria injected E) B and D are correct Note that the bacteria in all cases is S.Pneumonia 11  Which researcher(s) injected mice with candidate macromolecule enzymes plus dead S-bacteria to show that the transforming factor was nucleic acid (DNA)? A) Watson and Crick B) Hershey and Chase C) Griffith D) Avery, Macleod and McCarty 12 When Hershey and Chase differentially tagged the DNA and proteins of phages and allowed them to infect bacteria, what did the viral phages transfer to the bacteria? A. radioactive phosphorus 32P and sulfur 35S B. Radioactively labeled (35S) protein C. DNA labeled with 32P D. DNA labeled with 35S 13 Part 2. The Chemical Structure of Nucleic Acids 14  The Chemical Structure of Nucleic Acids Nucleic acids = DNA and RNA, both are polymers of ______________________ – Each nucleotide is composed of a 5-carbon sugar, 1- 3 phosphate group(s), and an organic nitrogenous base (all covalently linked)  nucleotides distinguished by the bases  dATP, dTTP, dCTP, dGTP Images from Brooker et al., 2007 15  5’ 3’  bases form hydrogen bonds with one another (weak)  held in a chain by a covalent phosphodiester bond (strong) 16 16  Chemical Nature of Nucleic Acids Pyrimidines - smaller bases – cytosine and thymine Purines - larger bases – adenine and guanine Chargaff’s rule  A = T and G = C  Implies “Base Pairing” http://www.dnai.org/a/index.html (click on “Code” in the menu bar) 17  In-Class Exercise Draw and label a nucleotide Which nitrogenous bases pair together? Which bases are larger (double rings), purines or pyrimidines? 18  Know Your Sugar – Pentose Numbering Be able to draw a ribose ring from memory Know how to label each carbon in this pentose 5’ Know what is attached 1’ 4’ to each carbon – 3’ 2’-OH in OH RNA understand the 3’ significance - of each part 5’ (–PO4 ) 3’ -OH Add next base to 3’ –OH during polymerization 19  Three-Dimensional Structure of DNA Wilkins and Franklin deduced DNA is a spiral structure using X-ray diffraction Watson and Crick deduced that DNA is an intertwined double helix – complementary base-pairing  purines pairing with pyrimidines  Applied Chargaff’s rules:  A = T -- they must pair  G = C -- they must pair  Antiparallel configuration 20  DNA Double Helix T A T C G C G C G Minor A T A Groove A T A Major G C G Groove Form fits function! Fidelity of sequence is passed to progeny 21 Three-Dimensional Structure of DNA 22  3’ -OH 5’ (–PO4) Add next base to 3’ –OH during 3’ -OH polymerization 23 23  At home/ In-Class Exercise Write a brief summary of the scientific discovery with which each scientist(s) is credited and the method used: – Wilkins and Franklin – Chargaff – Watson and Crick Draw a DNA double helix: label the bonds in the backbone and between bases, label the bases that pair, label the 3’ and 5’ end 24  Double Helix Sugar-P backbone Phosphodiester bonds (covalent, strong) Complimentary Base pairs Hydrogen Bonding: A - -T & C - - - G Anti-parallel strands 3’ and 5’ End (polarity) 25  Replication Each chain in the helix is a complimentary mirror image of the other. Because there are two DNA molecules in the double-helix, this is a clue to how it works... but how can it come apart and replicate? 3 models: – Conservative – Dispersive – Semiconservative 26  Meselon and Stahl showed that DNA replication occurred in a semi-conservative manner 27 27  Replication Process - Basics The two complimentary strands of double helix unwind & unzip: each serves as a template DNA polymerase III adds nucleotides to build new DNA strand. How does it know which nucleotides to add? Nucleotides are added according to complementary base pairing, H bonds One Direction of Synthesis: 5’  3’. In other words, a new daughter strand is made by adding new nucleotides 5’  3’ 28 DNA Replication Bidirectional Semiconservative 29 Replication Steps – first Learn the key components (enzymes) 30  Replication Process Replication origin (ori) - Replication of DNA begins at one or more sites 31  Replication Process Steps (enzyme) – key components Opening of the DNA double helix (helicase) – initiating replication and unwinding of the duplex at the replication origin – Creates replication fork 32  Replication Fork - Helicase *ATP hydrolysis* Powered by ATP, Helicase unwinds the DNA at the replication fork Also 33 note the ori, where replication begins 33  Replication Process - Steps (enzyme) Opening of the DNA double helix (helicase) – initiating replication and unwinding duplex at replication origin – SSB proteins (single stranded binding proteins): stabilizing single strands, prevents them from rejoining – relieving torque/tension created by the opening of the DNA helix. Works ahead of replication fork, avoids twisting of DNA. (gyrase or topoisomerase) 34  Helicase separates the parental DNA Topoisomerase (gyrase) removes torsional strain introduced by opening double helix SSBs 35 35 3-D structure DNA Polymerase (Beta) 36  Replication Process - Steps (enzyme) Assembling complementary strands (DNA polymerase III) – DNA Polymerase III –adds nucleotides to growing complementary DNA strands – Can only synthesize 5’ to 3’, must add to 3’ OH – requires a primer (brief sequence of RNA) – It reads/copies the parent/template from 3’  5’ – It adds nucleotides 5’  3’ on daughter strand Building a primer, made of RNA (primase) Removing the primer &replacing with DNA (DNA polymerase I) – DNA Pol I : cuts out RNA primer using its exonuclease activity; replaces the primer with DNA using its polymerization activity Joining fragments (ligase) 37 Primase (RNA polymerase) DNA Pol III - adds nucleotides Sliding DNA Clamp – Protein attached to DNA pol III, encircles DNA &Tethers DNA 38  Leading Strand - continuous Lagging Strand - discontinuous 39 40  DNA REPLICATION - steps Now you know the “key players”, learn the process, step by step….. 41  DNA Synthesis Leading strand 5’ to 3’ Direction of DNA Unwinding 5’ to 3’ Lagging strand 42  Replication Process - Steps Recall DNA polymerase III cannot link the first nucleotides in a newly synthesized strand, needs a primer: -Primase constructs an RNA primer DNA pol III adds nucleotides to 3’ end of the primer – Leading strand replicates toward replication fork. – Lagging strand elongates away from replication fork. =Okazaki fragments 43  1 2 44 44  3 Energy 45  Replication Process – enzymes DNA ligase attaches individual Okazaki fragments and also joins newly-synthesized DNA at the origins of replication Recall Helicase is continually separating the parental DNA Recall Topoisomerase (gyrase) is continually removing torsional strain ahead of the replication fork 46  4 -Excises RNA primer -Replaces primer w DNA 5 47 47 6 48 DNA Replication Fork – nucleotides added 49  Part 4. DNA Synthesis Errors, Telomeres, and DNA packaging Note: We will cover these topics in the context of a three-part case study in lectures. These are the associated notes, but the actual case study notes will not be posted on D2L 50  Errors in DNA replication and mechanisms of Correction Base-Pair mismatch = Wrong nucleotide added A) Proofreading: DNA Polymerase itself “proofreads” the newly made DNA. If it finds a mismatch, it reverses direction, excises the wrong nucleotide, adds the correct B.P., continues synthesis – Errors ~1 in 1,000,000 so have further scrutiny….. B) DNA Repair mechanisms: They scan the newly synthesized DNA and excise any mismatches that were missed by DNA Polymerase 51 Base-Pair mismatch = Wrong nucleotide added A) Proofreading: DNA Polymerase itself “proofreads” the newly made DNA. If it finds a mismatch, it reverses direction, excises the wrong nucleotide, adds the correct B.P., continues synthesis – Errors ~1 in 1,000,000 – Therefore have further scrutiny….. 52 53 If a mismatch eludes proofreading: B) DNA Repair mechanisms: They scan the newly synthesized DNA and excise any mismatches that were missed by DNA Polymerase It locates the distortion, removes it and surrounding nucleotides, DNA Pol fills the gaps, and ligase seals the nick 54 Telomeres 55  DNA polymerase cannot copy the tip of the DNA strand with a 3’ end – No place for upstream primer to be made – As a result of DNA not being copied, chromosomes get progressively shorter with each replication Telomerase prevents chromosome shortening How? – It attaches many copies of repeated DNA sequences to the ends of the chromosomes – Provides upstream site for RNA primer to attach 56 Image from Brooker et al., 2007  Telomeres Telomere: telo = end mere = segment – Region of non-coding repeat (100-1000s of repeats) DNA sequences found at the end of chromosomes; protects genes found near Chrs ends by providing “buffer zone” Telomerase: enzyme that adds DNA to the end of chromosomes to maintain the “buffer zone” of telomeres; it has its own RNA template – The repeats are TTAGGG in humans – Most somatic cells in multi-cellular organisms do not contain telomerase, they undergo mitosis without telomerase 57 3’Overhang – no where to put a primer so DNA Pol III can NOT add nucleotides Telomerase binds at 3’ end/ telomere – It adds a telomere DNA repeat to the 3’ end Telomerase slides along to newly formed 3’ end 58 -Telomerase continues to add more telomere DNA repeats to extend 3’ end -The new telomere end has now provided a comp.strand for Primase to add a primer -DNA Polymerase adds nucleotides to 3’ OH of primer – filling region that otherwise would not have been replicated -Still have 3’ overhang, but it’s just telomere repeats 59  Telomeres, Aging, and Cancer - Research Application Hayflick’s limit: cultured human cells can only divide a certain number of times before dying – cells taken from infants – 80 divisions – older persons – 10 to 20 (age dependent) Shortening of telomeres correlates with loss of vitality;most somatic cells do not have telomerase – germ cells, stem cells – combat senescence using telomerase Cancerous cells often have restored telomerase activity  do NOT show Hayflick’s limit in culture 60  DNA is packaged into Chromosomes Chromatin – DNA plus associated proteins Nucleosome – DNA wrapped around 8 histones Separate During Mitosis/Meiosis II 61 Review – DNA structure and DNA Synthesis 62 63 Okazaki fragments form on the _______ strand and they are linked covalently by ________ A. Template strand, Primase B. Leading strand, ligase C. Lagging strand, ligase D. Leading strand, DNA Pol I E. Lagging strand, Primase 64 64 DNA polymerase III can only add nucleotides to an existing chain, therefore _______ is required A. helicase B. A Primer, brief RNA sequence C. DNA polymerase I D. A Primer, brief DNA sequence 65  In-Class Exercise – Enzyme List Helicase - Opens DNA double helix, initiating replication and unwinding duplex SSBP - stabilizing single strands (not enzyme) Primase - Building a primer of RNA Topoisomerase - relieves torque, prevents twisting of DNA DNA polymerase III - Assembles complementary strands by adding DNA nucleotides DNA polymerase I - Removing RNA primer and replacing it with DNA Ligase - Joining Okazaki DNA fragments 66 In Class Exercise Number the following events in the order in which they occur in DNA replication of the lagging strand: ___ DNA ligase joins the Okazaki fragments ____ 1 replication proteins bind to and activate the origin of replication (ori) ____Primase synthesizes an RNA primer ____Helicase unwinds the double helix ____ DNA pol I replaces the RNA primer with DNA ____ SSBPs stabilize and hold the single strands apart ____ DNA pol III adds DNA nucleotides to the primer 67 If one strand of a DNA molecule has the base sequence 5'ATTGCAT3', its complementary strand will have the sequence A. 3'ATTGCAT5' B. 3'GCCATGC5' C. 3'CGGTACG5' D. 3'TAACGTA5' E. Can't be determined from the information given 68 If one strand of a DNA molecule has the base sequence 5'AATACGG3', its complementary strand will have the sequence: A. 5'CCGTATT3' B. 5'AATACGG3' C. 5'GGCATAA3' D. 5'TTATGCC3' E. Can't be determined from the information given 69  DNA and Replication 1. What is the monomer unit of DNA? 2. List the 4 Nitrogen bases. To which Carbon do they attach? 3. To which end of DNA is a new nucleotide added? 4. What classification of chemical reaction is DNA synthesis? 5. The Phosphate of an incoming nucleotide is attached to the __’C 6. To which end are new nucleotides added? 7. What is the direction of chain growth? 8. Types of Bonds: btw nucleotides? 9. DNA has a ____-_____ backbone 70  DNA Replication Which enzyme adds new nucleotides? What are the small blue RNA pieces called? Which enzyme adds these primers? Is the top strand leading or lagging strand? Which enzyme joins together the existing Okazaki DNA fragments on the lagging strand? Which enzyme relieves tension and torque? 71  What is the name of the repeat sequences at the Chrs ends? Is telomerase found in all cells? Which is the lagging strand in this diagram? 72  DNA – Research Summary Discovery that DNA was the genetic material, heritable: Griffith, 1928: Mice infected w R- or S- strains of pneumonia. R-strains could be transformed to ‘S’ by transforming principle, which therefore must be the genetic material. Avery, McCarty, and Macleod, 1944: In vitro, investigated which component of S-strain extract was transforming principle  DNA Hershey and Chase 1952: Radio-Labelled protein coat & DNA of Phages. Confirmed DNA was genetic material Structure of DNA: Wilkins and Franklin: conducted Xray diffraction experiments on DNA to deduce DNA was a helical spiral structure Chargaff: showed A : T and C : G occur in equal proportions in DNA Watson and Crick, 1953: constructed a model based on above experimental data to determine the double helix model of DNA Meselson and Stahl: used DNA grown on 15N and 15N mediums to show DNA replication occurs via semi-conservative method 73