Biology 135A DNA Structure and Replication PDF

Biology 135A DNA Structure and Replication (Ch. 16) Rajab Outline Discovery of DNA as the genetic material of life DNA structure DNA replication DNA Proofreading and repair Telomeres DNA packaging ...

Biology 135A DNA Structure and Replication (Ch. 16) Rajab Outline Discovery of DNA as the genetic material of life DNA structure DNA replication DNA Proofreading and repair Telomeres DNA packaging  the r cells become s cells after taking whatever the virulent - cause s cells are avirulent Is the genetic matter protein or infection giving (they broke open DNA??? after dead and their DNA as the genetic material: genetic material is – DNA vs. proteins taken by the living r 1928 Frederick Griffith— cells) —> tranformative factor —> Streptococcus pneumoniae genetic materials – S (have protective capsule) and R (no protective capsule) strains and transformation – Experimental model to test DNA vs protein hypothesis no capsule = body can kill them What was transforming substance? ***need to know experiments 1944 Avery et al. → purified DNA is Griffith’s transforming factor their question?: what is injected into the host —> protien or dna? 1952 Alfred Hershey and Martha Chase—Bacteriophages – T2 phage made of protein and lytic cycle DNA – Grew T2 w/ bacteria & either radioactive 35S (proteins labeled) or radioactive 32P (DNA labeled) – Radioactive T2 now mixed with new bacteria, spun, and centrifuged → is radioactivity inside bacteria or outside bacteria? Hershey/Chase Expt: Is DNA or protein the genetic material of phage T2? RESULT: Phage proteins remained outside the bacterial cells during infection, while phage DNA entered the cells. When cultured, bacterial cells with radioactive phage DNA released new phages with some radioactive phosphorus. CONCLUSION: DNA, not protein, functions as the T2 phage’s genetic material. STRUCTURE OF DNA What was known about DNA before 1950: – Nucleotides of sugar, phosphate and base [A, G (purines), T, or C (pyrimidines)] – 1947 Erwin Chargaff: ratio of A, T, C, and G differs between different species A and T occur in same 1:1 ratio and C & G occur in same 1:1 ratio DNA composition varies from species to species.  The structure of DNA must allow the four functions of DNA – Store info sequence of bases when separated each – Precisely replicated strand can act as a template for making new DNA sequence means – Express a phenotype something & can be read & made into some product (usually RNA molecule that then makes s protien) – Change important for evolution otherwise there would only be one species occurs during mutations/errors of replication or recombination  1953 Watson and Crick — DNA is a double helix – based on X-ray crystallography of Rosalind Franklin/Maurice Wilkens) and modeling: What won the Nobel Prize? antiparallel, right-handed 1 turn = 3.4 nm distance between any two adjacent nucleotides = 0.34 nm regulatory protiens major and minor grooves that determine structure suggests replication method whether a gene is on or off A binds with T and C with G binds to the major grooves DNA Replication The basic idea: one strand acts as a template for making a new strand Four requirements for replication: – A template is needed → each strand is template for complimentary base-pairing – Building blocks needed → 4 dNTPs – Enzyme needed → DNA polymerase – Energy needed → phosphoanhydride bonds of dNTPs Three alternative models of DNA replication Mechanisms of replication: – 3 possible models: Conservative Semiconservative Mixed or dispersive – Which one was correct?  Matthew Meselson and Frank Stahl (Cal Tech) 20 min 40 min 60 min - 3 replications - 3 generations density gradient centrifugation= method to tell apart heavy vs light DNA CONCLUSION DNA replication follows the semiconservative model  Three major steps: 1. DNA is locally denatured at origins of replication 2. New nucleotides are linked by covalent bonding: free 3' OH reacts with one of the phosphates of either dATP, dGTP, dCTP, or dTTP and pyrophosphate is released PPi → 2Pi + free energy 3. Strand grows 5’→3' – DNA replicates in both directions from origin → replication fork at each end. – Proteins of rep. complex: 1. DNA helicase: uses energy of ATP to separate strands + topoisomerase unwinds supercoils 2. Single-strand binding proteins: maintain single-strands 3. RNA primase: lays down an RNA primer because DNA polymerase can’t start a polynucleotide, only elongate it.  4. DNA pol III in bacteria extends the new DNA strands 5. DNA pol I→ repair 6. DNA ligase Steps of Replication melted DNA has two templates: – template for leading strand: is read 3’ → 5' continuously only one primer needed  template for lagging strand: – is read 3’→5' also, but discontinuously – many primers and DNA fragments-Okazaki fragments  DNA pol I removes old RNA primer and replaces it with DNA Nicks between fragments joined by ligase https://www.youtube.com/watch?v=TNKWgcFPHqw  Telomeres Telomeres are the ends of eukaryotic chromosomes and buffer chromosomes from loss of information Are rich in TTAGGG (in humans) protect chromosome from unraveling, buffer it from loss of replication no genes to be turned on in telomeres, you can silence certain genes by putting them in telomeres? we age because of telomere shorterning (short fragments after replication) & oxidative stress that damages cells  Telomere end replication problem – 3’ ends of lagging strand template are not replicated (5’ end of lagging strand not made) → End replication problem: continued shortening of DNA strands www.youtube.com/watch?v=eCfcl0h-F4k mere Talk Yale Telomere Talk https://www.youtube.com/watch?v=eCfcl0h-F4k https://youtu.be/2NS0jBPurWQ Werner syndrome causes premature aging → mutation in WRN protein of cap structure SCIENCE! Dr. Elizabeth Blackburn: Won the Nobel Prize for the SCIENCE co-discovery of telomerase. Prior to this, she was in the FICTION! group that discovered AKA BF → telomeres. She was a professor/researcher at UC (Bull Feces!) San Francisco and past president of the prestigious Salk Institute. Watch this TED talk by Dr. Elizabeth Blackburn. https://youtu.be/2wseM6wWd 74 Another video to check out- Yale Telomere Talk: https://www.youtube.com/watc h?v=eCfcl0h-F4k Proofreading & Repair: Replication in E. coli is fast (1000 b.p./sec) with errors in fewer than 1 in 104 bases, but mutation rate is 1 in 109 nucleotides. DNA is subject to errors of replication & environmental damage – there are at least 3 repair mechanisms: 1. Proofreading mechanism of polymerase→ corrects errors as made; fixing in the moment 2. Mismatch repair → scans DNA after it is made and corrects mismatches → deamination of C to T is common the incorrect base is recognized because it is not methylated yet. How might repair enzymes know when there has been a mismatch? 3. Excision repair → removes UV or chemically damaged bases → thymidine dimers xeroderma pigmentosum disorder Mutations in DNA repair proteins can result in human diseases Unprotected sun exposure causes 9-4_xp_child_in_suit lesions and skin cancer Xeroderma Pigmentosum Packaging DNA octomer https://youtu.be/gbSIBhFwQ4s Chimerism in Humans