Pelligrini Lectures: Genome

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Pelligrini Lectures: Genome
2012-05-11 17:01:29
genome dna synthesis repair replication polymerase molecules chemicals

5 lectures: DNA, synthesis, genomes, working with DNA, DNA mutation and repair
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  1. oriC
    Bacterial origin of replication
  2. DNA A
    Melts bacterial DNA at Origin of replication
  3. DNA B
    bacterial DNA helicase, creates replication forks
  4. DNA Pol III
    Bacterial main DNA polymerase
  5. DNA Pol I
    Bacterial polymerase, 5-3 RNA exonuclease activity and 3-5 proofreading activity
  6. ORC
    6 protein complex, recognises eukaryotic origins of replication and recruits DNA helicase
  7. MCM
    eukaryotic helicase for replication, 6 subunits
  8. Cdc6, Cdt1
    Licensing factors for ORC to recruit MCM (2)
  9. Dna C
    Bacterial protein, helps load helicase to origin
  10. RPA
    Single stranded binding protein in eukaryotic cells
  11. DNA Pol alpha, delta, epsilon
    Eukaryotic DNA polymerases
  12. PCNA
    Sliding clamp in eukaryotic cells
  13. RFC
    Eukaryotic clamp loader
  14. gamma-complex
    bacterial clamp loader
  15. ATP dependent DNA ligase
    eukaryotic DNA ligase
  16. NAD dependent DNA ligase
    bacterial DNA ligase
  17. RNaseH
    eukaryotic: removes primer of RNA paired to DNA, leaving small flap
  18. Fen1
    Cuts off flap of primer in eukaryotic cells
  19. Topoisomerase
    Untangles DNA during replication. Type I cuts a single strand, Type II cuts a duplex, passing one under the other so positive supercoiling is reduced.
  20. Etoposide, doxorubicin
    Inhibit topoisomerase II (2)
  21. camptothecin
    inhibits toposomerase I
  22. Heterochromatin
    Densely packed DNA, not transcribed, replicated late is S phase, no meiotic recombination
  23. Euchromatin
    transcribed DNA - not as tightly packed
  24. Barr bodies
    Inactivated X chromosomes, seen at side of cell
  25. Telomerase
    self-templating reverse transcriptase (protein and RNA template components)
  26. Hayflick's limit
    the amount of times somatic cells (20-30), depending on the length of their telomeres
  27. T loop
    structure at end of telomere, protects from recombination/degradation/end-to-end fusion
  28. TTAGGG
    Repeating unit (About 1000 times) in telomeres
  29. Retrotransposons
    Transposition via RNA. Can be conserative of replicative
  30. Transposase
    Enzyme that moves transposons around the genome
  31. RAG recombinases
    recombination activation gene - mediate antibody diversity
  32. 4 types of mobile genetic elements
    • - LINEs (long interspersed nuclear elements)
    • - SINEs (short ...) (inc Alu!)
    • - Retrovirus like elements
    • - DNA transposon fossils
  33. 4 classes of repetitive sequences
    • - 3% Simple sequence repeats, <100bp, CACACACACACA
    • - 5% Segmental duplications, 10-300kb
    • - 45% Transposon-derived repeats
    • - Inactive copies of partially retrotransposed cellular genes
  34. EcoRV
    Restriction endonuclease, makes blunt ended DNA molecules
  35. EcoRI
    Restriction endonuclease, makes sticky ended DNA molecules (--TTAA)
  36. Dye used in Southern Blotting, intercalates between DNA
    ethidium bromide
  37. PCR temperatures
    • - >90 degrees: melting
    • - 55-60 degrees: annealing primers
    • -72 degrees: extending
  38. FISH
    fluorescent in situ hybridisation - can look for chromosomal rearrangements, gene copy number in cancer cells
  39. Transition
    Purine swapped for purine, or pyrimidine swapped for pyrimidine
  40. Transversion
    Purine swapped for pyrimidine or vice versa
  41. 5 types of DNA replication errors
    • - Base mismatches
    • - Deletions
    • - Insertions
    • - Incomplete replication
    • - Breaks
  42. 3 types of single strand DNA break repair
    • - Base excision repair
    • - Nucleotide excision repair
    • - Mismatch repair
  43. 3 examples of base excision repair
    • Cyclobutyl ring: UV causes dimersation of two Ts, (obstructs replication/transcription) DNA photolyase photoreactivates it back
    • Tautomeric shift cytosine -> uracil: via spontaneous deamination/nitrite treatment - Uracil DNA glycosylase (UDG) cleaves the U, nuclease (AP endonuclease (bac)/pol beta (humans)) cleaves backbone, DNA pol (1/beta) fills in gap
    • 8-oxoG: flipped, and recognised as T (specific DNA glycosylase removes 8-oxoG)
  44. Example of nucleotide excision repair
    UV induced thymidine dimer (eukaryote): DNA unwound around lesion, strand cut up/down stream, gap filled by DNA pol and sealed by DNA ligase
  45. 2 types of nucleotide excision repair
    • Global genomic NER
    • Transcription coupled repair: RNA pol II holoenzyme (transcribed genes repaired faster)
  46. Mismatch repair detection pro/eukaryotic
    • Prokaryotic: hemimethylated strand
    • Eukaryotic: use nicks
  47. 2 types of double strand repair
    • NHEJ: direct rejoining of ends, low fidelity - can lose DNA. Important in resting, G1 and G0.
    • Homologous recombination: uses sister chromatid to find matching sequences. Also used to restart stalled/broken DNA replication forks. Also for gene targeting in embryonic stem cells.
  48. RecA (bacteria), RAD51 (eukaryotes)
    Recognise single stranded 3' end in HR, scans genome for identical strands and directs strand invasion. Eukaryotic/prokaryotic forms.
  49. RuvAB
    Facilitates Holliday junction branch migration in bacteria
  50. VDJ recombination
    antibody diversity generation, using HR. diversity increased by enzymes eg terminal transderase (adds extra nucleotides at junction point... sloppily)