Requirements for the Genetic Material

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Lenard Watkins
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1 Requirements for the Genetic Material 1. Replication Reproduced and transmitted faithfully from cell to cell-generation to generation. 2. Information Storage Biologically useful information in a stable form. 3. Expression of Information Express itself: Other biologically important molecules, and ultimately cells and organisms, will be produced and maintained. 4. Variation (by mutation) Capable of variation: some change is required

2 REPLICATION OF DNA

3 Replication of DNA What is the mode of DNA replication and how was that determined? Fig. 10-1

4 Replication of DNA: 3 possible models Fig. 10-2

5 Meselson-Stahl Experiment Grew E. coli in medium containing ammonium chloride ( 15 NH 4 Cl) as sole N source 15 N = heavy but nonradioactive isotope After multiple generations, all N-bases of DNA contain 15 N Extracted DNA and centrifuged in density gradient (CsCl) Grew 15 N labelled cells in regular medium ( 14 NH 4 Cl) Collected samples after 1st, 2nd and 3rd generations Extracted DNA and centrifuged in density gradient (CsCl) Compared banding patterns observed in density gradients

6 Fig Meselson-Stahl Experiment

7 In the Meselson-Stahl experiment, if DNA is replicated conservatively, then after 2 generations of replication there would be A) one old band and one new band B) one hybrid band C) one hybrid band and one new band D) one hybrid band and one old band E) one old band, one hybrid band, and one new band

8 DNA Replication Bacterial & eukaryotic models Similar process: Initiation Elongation Termination Differences due to differences in DNA structure and complexity of DNA coiling Circular vs. linear DNA Single vs. multiple origins of replication Absence or presence of nucleosomes

9 DNA Replication in Bacteria Fig. 10-6

(single-stranded binding proteins) Problem Unwinding creates

10 DNA Replication Unwind & denature double helix Helicases Unwind, open & stabilize helix DnaA, DnaB, DnaC Stabilized by SSB s (single-stranded binding proteins) Problem Unwinding creates supercoiling causes torsional stress Relieved by DNA gyrase SS or DS nicks Fig. 10-9

11 DNA Replication Initiate synthesis RNA primase (RNA polymerase) adds an RNA primer ~5-15 nucleotides Fig. 10-9

12 The role of the DnaA protein in bacterial DNA replication is to A) prevent renaturation of the two DNA strands during replication B) synthesize and RNA primer C) relieve the tension of supercoiling D) initially unwind the DNA double helix E) detect replication errors

13 Chain elongation DNA pol III DNA Replication Requires free 3 -OH to bind Adds nucleotides 5 3 Fig. 10-9

14 Fig. 10-7

15 Elongation Elongation is... Simultaneous Both strands at once Bidirectional Strands are antiparallel Continuous & discontinuous processes Leading strand (continuous) Lagging strand (discontinuous Okazaki fragments Fig simultaneous synthesis

16 Okazaki fragments are a consequence of A) the inability of DNA polymerase to initiate a new DNA strand B) the inability of DNA polymerase to correct replication errors C) random strand breakage due to supercoiling D) mutations in the gene for DNA ligase E) the inability of the DNA polymerase to synthesize in the 3-5 direction

17 DNA Replication Primers removed and lagging strand gaps filled DNA pol I Cleaves out RNA primer and replaces with DNA Has both 5 activities DNA ligase 3 exonuclease and polymerase Forms final phosphodiester bond to fill gap

18 Chain Elongation Enabling of concurrent synthesis DNA pol III is a dimer Loop forms to keep antiparallel template strands effectively 3 5 relative to DNA pol III bp Fig

19 Proofreading Critical that new strand is exact complement of template DNA pol I & III have 3 activity 5 exonuclease Recognition & replacement of mismatches during elongation back-up and replace mismatched bases DNA pol II Active in DNA repair to external damage (i.e., UV light)

20 Polymerase Comparison

21 Bacterial DNA Replication Summary dna replication Fig

22 All known bacterial DNA polymerases A) can initiate DNA chain synthesis B) have 5 to 3 polymerization activity C) have 5 to 3 exonuclease activity D) have 3 to 5 polymerization activity E) all of the above

23 Replication of Eukaryotic DNA Problems Larger genomes Eukaryotic DNA pols are slower Nucleosomes Linear chromosomes

24 Replication of Eukaryotic DNA Solutions Multiple origins of replication Yeast : replicons Mammals: up to 25,000 replicons More types (14) of polymerases Different activities / operate under different conditions More polymerase molecules per cell E. coli ~400/cell Homo sapiens ~50,000/cell

25 Comparison of Replication Rates E. coli 4.7 kb ~20-40 min Drosophila 120 kb ~ 3 min Homo sapiens 3300 kb ~ 7 hrs

Replication of Eukaryotic DNA Replication problems at ends (telomeres) of linear chromosomes RNA primer at terminal end Once removed, no 3 -OH available for

26 Replication of Eukaryotic DNA Replication problems at ends (telomeres) of linear chromosomes RNA primer at terminal end Once removed, no 3 -OH available for addition of DNA nucleotides Can lead to telomere shortening (cellular clock) Fetal tissue culture cells divisions max. Adult cells divisions max Fig

27 Telomeres Repeated sequences Tetrahymena a protozoan TTGGGG tandem repeats Overhang on G-rich strand of bases G-quartets form loops on ends of chromosomes Fig

28 Telomerase A ribonucleoprotein Contains RNA (-AACCCCAAC-) Recognizes telomeric sequence and adds repeats RNA primase DNA pol I & ligase Typically low activity in somatic cells; high activity in cancerous cells Fig

29 Telomerase A) is a ribonucleoprotein B) has reverse transcriptase activity C) adds short tandem repeats to the ends of chromosomes during DNA replication D) functions to replicate DNA at the ends of linear chromosomes E) all of the above

30 Recombination (Crossing Over) 1. Homologs pair and synapses form 2. Endonucleases nick DNA at adjacent sites on both homologs SS 3. Ends are displaced and pair with homologous sequence on other duplex Fig

31 Recombination 4. Ligase seals nicks Creates heteroduplex 5. Branches migrate H-bonds unzip and form complementary bonds with other duplex 6. Duplexes separate & rotate Chiasmata Fig

32 Recombination 7. Endonucleases nick opposite sides of chi form 8. Homologs are ligated and separated Fig

33 Recombination A) is a ribonucleoprotein B) has reverse transcriptase activity C) adds short tandem repeats to the ends of chromosomes during DNA replication D) functions to replicate DNA at the ends of linear chromosomes E) all of the above

34 Gene Conversion Mismatch after crossover can result in mutation Fig