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MOLECULAR GENETICS

MOLECULAR GENETICS. DNA SYNTHESIS. OBJECTIVES. Understand the process of DNA replication Be familiar with the kinds of errors that may occur during replication of DNA Understand how protein structure and function are affected by genetic mistakes. FUNCTIONS OF DNA. Functions: DNA Synthesis

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MOLECULAR GENETICS

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  1. MOLECULAR GENETICS DNA SYNTHESIS

  2. OBJECTIVES • Understand the process of DNA replication • Be familiar with the kinds of errors that may occur during replication of DNA • Understand how protein structure and function are affected by genetic mistakes

  3. FUNCTIONS OF DNA Functions: • DNA Synthesis • DNA makes more DNA • Protein Synthesis • TranscriptionTranslation

  4. DNA SYNTHESIS When In the Cell Cycle Is The Amount of DNA Doubled? Sister chromatids

  5. Cell Before S-phase Nucleus After s-phase

  6. MODELS OF DNA REPLICATION • Three Models of DNA Replication: • Semi-conservative Replication • Conservative Replication • Dispersive Replication

  7. MODELS OF DNA REPLICATION Hypothesis A Hypothesis B Hypothesis C Semi-conservative Replication Conservative Replication Dispersive Replication

  8. MODELS OF DNA REPLICATION Two double helices Double helix Protein that copies DNA If DNA replication is semi conservative, each new double helix has one original and one new strand

  9. MODELS OF DNA REPLICATION • Meselson & Stahl Experiment: • Methods: • 15N was fed to growing E. coli cells to • mark DNA • Then cells switched to 14N • Results: • After 2 generations, ½ samples had low density & other ½ had intermediate density • Conclusions: • New DNA composed of: • Entirely 14N • One 15N strand and one 14Nstrand • DNA replication issemi conservative

  10. DNA SYNTHESIS IS SEMI-CONSERVATIVE • DNA replication is semi-conservative in that each new DNA molecule incorporates an old strand that serves as a template

  11. MECHANICS OF DNA SYNTHESIS • Replication Fork: • Place where double stranded DNA opens to form a “bubble” • Origins of Replication: • Regions on the DNA where synthesis begins

  12. MECHANICS OF DNA SYNTHESIS • DNA Synthesis Requires An Enzyme (Kornberg, 1950s)

  13. MECHANICS OF DNA SYNTHESIS • DNA Polymerase: • Forms new strand of DNA • Complementary to template

  14. MECHANICS OF DNA SYNTHESIS • But first, DNA must be “primed”

  15. MECHANICS OF DNA SYNTHESIS Primase: • Enzyme that adds initial nucleotides (RNA primer) to template strand • RNA primer later replaced with DNA

  16. MECHANICS OF DNA SYNTHESIS DNA Synthesis: • Initiated when DNA Polymerase (Pol III) binds to RNA primer • Pol III adds nucleosidesto growing new strand • Nucleoside triphosphate (dNTPs) • Nucleotide with 3 phosphates • Four kinds (depends on base): • dATP • dTTP • dGTP • dCTP • Numbering Carbons P P P base 5 sugar 1 4 2 3

  17. MECHANICS OF DNA SYNTHESIS DNA Polymerase (Pol III): • Catalyzes formation of phosphodiester bonds between nucleoside & nucleotide • Breaking bonds b/w phosphates provides energy for snythesis rxn P P P CH2 5' Base O 3' OH Structure of nucleoside (dNTPs)

  18. DNA Polymerase adds nucleosides to 3’ end of growing DNA strand Free OH New DNA grows from 5’3’ MECHANICS OF DNA SYNTHESIS 5’ 3’ end Next nucleoside added to this end!! OH

  19. MECHANICS OF DNA SYNTHESIS Growing Strand Of DNA 5’ 5’ base 5’ + sugar 3’ Next nucleoside added to this end!! P P + H2O 3’ OH 3’ OH

  20. MECHANICS OF DNA SYNTHESIS NEW OLD DNA Strands Are Antiparallel!

  21. MECHANICS OF DNA SYNTHESIS • New DNA is synthesized in the 5’  3’ direction • Nucleosides added to 3’ end of growing strand

  22. MECHANICS OF DNA SYNTHESIS • Leading Strand: • New growing strand (continuous) that follows replication fork • Lagging Strand: • Strand that grows (discontinuous) in direction away from fork

  23. FORMATION OF THE LEADING STRAND 3' DNA polymerase III 5' 5' Newly synthesized leading strand 3' 5' Replication fork DNA IS UNWINDING & OPENING IN THIS DIRECTION

  24. MECHANICS OF DNA SYNTHESIS Lagging Strand: • Synthesized in short pieces called Okazaki fragments, each with their own primer • Fragments later joined together by DNAligase

  25. FORMATION OF LAGGING STRAND 3' 5' Lagging strands 5' 3' 3' DNA polymerase III 5' 3' 5' Okazaki fragments 5' 3' DNA polymerase III beginning synthesis of new fragment 3' Gap 5'

  26. ROLE OF PROTEINS IN DNA SYNTHESIS Proteins In DNA Synthesis: • DNA Polymerase • Many types • Primase: • Synthesizes RNA primer • Helicase: • Unwinds strands of DNA • Single-Strand Binding Proteins: • Keep original complimentary strands separated • Ligase • Links O. Fragments into continuous strand

  27. DNA REPLICATION 3 Pol III synthesizes leading strand 1 Helicase opens helix Primase synthesizes RNA primer 2 4 Pol I excises RNA primer; fills gap 5 6 Pol III elongates primer; produces Okazaki fragment DNA ligase links Okazaki fragments to form continuous strand

  28. MUTATION Mutation: • Any change in an organism’s genome • Often occurs during DNA replication A G

  29. AN UNCORRECTED GENETIC ERROR Mutations can occur during DNA synthesis A A C T G G C Wild type T T G A C C G A A C T G G C A A C T A G C MUTANT 3' 5' T T G A T C G T T G A T C G A A C T G G C DNA replication DNA replication T T G A C C G A A C T G G A A C T G G C C 5' 3' Wild type Parental DNA T T G A C C G T T G A C C G First generation progeny A A C T G G C Wild type T T G A C C G Second generation progeny

  30. MUTATION UV Light • Mutations Can Also Result From: • Mistakes in mRNA synthesis (transcription) • Exposure to chemicals or radiation • Errors in meiosis • Nondisjunction • Breaks in chromosome

  31. MUTATION Types of Mutations: • Base-pair Substitutions: • Replacement of one or more nucleotides with another • Silent • Missense • Nonsense • Base-pair Insertion or Deletion: • Change in number of nucleotide pairs • Can result in frame shifting • Silent • Missense • Nonsense

  32. MUTATION Mutations can be Deleterious, Beneficial, or Silent: • In individuals are usually deleterious • Change in DNA sequence often produces protein(s) of abnormal form & function • Cause disease and death • In populations, they are a source of genetic diversity • Allow evolution to occur

  33. MUTATIONS Not all mutations are deleterious…. EX: Silent mutation

  34. MUTATION • Not all mutations are heritable! • Depends on whether mutation is present in gametes or in somatic cells only

  35. MOST MUTATIONS ARE DELETERIOUS, SOME ARE HERITABLE: SICKLE CELL ANEMIA Phenotypes: Start of coding sequence CAC GTG GAC TGA GGA CTC CTC DNA sequence GTG CAC CTG ACT CCT GAG GAG Normal Amino acid sequence Normal red blood cells Histidine Threonine Glutamic acid Glutamic acid Valine Leucine Proline CAC GTG GAC TGA GGA CAC CTC DNA sequence GTG CAC CTG ACT CCT GTG GAG Mutant Amino acid sequence Sickled red blood cells Threonine Histidine Glutamic acid Leucine Valine Valine Proline Some DNA point mutations lead to a different amino acid sequence.

  36. GENETIC REPAIR MECHANISMS • Mismatch Repair: • Mechanism by which mismatched nucleotides are repaired by DNA Polymerase during DNA synthesis

  37. MISMATCH REPAIR 3' 5' Mismatched bases. A T G T C C T C G C A C A G G DNA polymerase III “proofreads” and corrects point mutations during DNA replication. G 5' OH 3' 5' Polymerase III can repair mismatches. 3' A T G T C C T C G C A C A G G 5' OH 3' T G OH

  38. GENETIC REPAIR MECHANISMS Excision Repair Systems: • Consists of coordinated groups of molecules • Excise stretch of single-stranded DNA, around damaged site • Resynthesize new strand based on intact, complementary strand

  39. MISMATCH REPAIR IN PROKARYOTES Methyl group on template DNA strand Mismatch

  40. METHYLATION-DIRECTED MISMATCHED BASE REPAIR 1. Where a mismatch occurs, the incorrect base occurs on the unmethylated strand. Mismatch 2. Enzymes detect mismatch and nick unmethylated strand 3. DNA polymerase I excises nucleotides on unmethylated strand. 4. DNA polymerase I fills in gap in 5‘ 3' direction. 5. DNA ligase links new and old nucleotides. Repaired Mismatch

  41. DEFECTS IN GENETIC REPAIR MECHANISMS • Defects in genes responsible for excision repair are frequently associated with cancer • Xeroderma pigmentosum (“XP”) • Individuals are 1000 – 2000 x as likely to get skin cancer Vulnerability of Cells To UV Light Normal cells % Cell Survival Cells from XP Patients Level UV Light

  42. DEFECTS IN GENETIC REPAIR MECHANISMS XP CELLS HAVE LITTLE OR NOABILITY TO REPAIR DAMAGE 60 50 Damaged DNA is repaired in normal individuals 40 Amount of radioactive thymidine incorporated (counts per minute) 30 20 Repair is defective in XP patients 10 0 Dose of UV light

  43. UV-induced thymine dimers caused DNA to kink H P O P H O CH2 CH2 DNA strand with adjacent thymine bases Thymine O O O O UV light H CH3 Thymine Dimer H CH3 P H P O O H Kink O CH2 CH2 O O Thymine O H CH3 H CH3 P P

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