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Biochem LEC 1-3

  1. fundamental structure of DNA molecule
    • 10.5 bp/turn
    • 0.34 nm rise/bp
  2. Is most cellular DNA under or overwound?
    underwound
  3. affects of underwinding
    induces negative supercoiling, decreases LK
  4. affects of overwinding
    induces positive supercoiling, increases LK
  5. linking number formula
    twist (T) + writhe (W)
  6. Bacterial Type I topoisomerase
    • relaxes negative supercoils
    • change LK in increments of 1
  7. Bacterial Type II topoisomerases
    • change increments of 2 
    • topo II(makes negative supercoils)
  8. Bacterial DNA gyrase(6)
    • type II
    • change LK by 2
    • ATPase
    • generates negative supercoils and relaxes negative supercoils
    • has open and closed conformation
    • catalyzes a double strand break
  9. Eukaryotic Topo II
    • Topo II relaxes both + and - supercoils
    • it cannot induce negative supercoils
  10. how is DNA supercoiled
    chromatin assembly in negative solenoid supercoil (forms nucleosomes)
  11. Meselson-Stahl experiment
    showed DNA was semi-conservative using heavy labeled nitrogen
  12. enzymes involved in DNA replication
    • Pol 1- used for DNA repair
    • Pol III- used for DNA replication in E.coli
    • DNA Ligase- used to repair nicks
  13. common properties of DNA polymerase
    • needs a primer with 3' OH 
    • driving force: H-bonding and base-stacking interactions
    • two-metal ion mechanism that supports the α 3 'OH attack of the incoming nucleotide alpha-P
  14. properties of DNA Polymerase I
    • low processivity
    • catalyzes both polymerization and hydrolysis
    • 3' to 5' exonuclease: enhances fidelity
    • 5' to 3' exonuclease: functions in RNA primer removal, DNA repair
  15. properties of polymerase III
    • primary POL involved in replication in E. coli
    • high processivity factor
    • consists of 10 different subunits
  16. how are RNA segments synthesized?
    primosome
  17. Eukaryotic DNA Replication enzymes
    • primase/Polα: function to synthesized RNA-DNA primers for replication (low processivity)
    • Pol δ and PCNA: function in lagging strand
    • Pol ε and PCNA: function in leading strand
  18. Function/structure of PCNA
    • homotrimer and functions like the bacterial III beta subunit 
    • different amino acid
  19. Mechanism of Reverse transcriptase (RT)
    • t-RNA primer base paired to viral genome
    • forms a DNA-RNA hybrid
    • RNase H breaks apart RNA strand
    • DNA Pol adds DNA to double stranded DNA
  20. mechanism of replication initiation in E. coli
    • -DnaA forms a right handed (positive) helical filament
    • -bends oriC DNA promoting unwinding
    • -negative supercoiling in A::T rich repeats(strand separation)
  21. regulation of replication initiation and chromosome paritioning
    • Fully methylated oriC binds DnaA and initiates replication
    • Hemimethylated oriC- cannot reinitiate
    • Dam methylase-methylates new oriC strand and initiation of replication
  22. Function of DnaA
    • uses ATP
    • binds to the Ori to form a positive(right-hand) supercoil 
    • results in negative supercoiling in adjacent A::T rich repeats
    • promotes strand separation
  23. function of ORC
    • origin recognition complex protein in yeast
    • atpase
    • promotes formation of right-handed DNA helix
    • it's bound to ori DNA throughout cell cycle
    • hexameric complex
  24. mechanism of cell cycle control in eukaryotic DNA replication initiation
    • Cdc6p binds to ORC (early G1)
    • MCM2-7 binds to the complex (helicase) and activates replication at G1/S phase
    • Cdc6p is phosphorylated and degradated (CDK activity is high)
    • ORC and helicase are phosphorylated and replicated
Author
Sheilaj
ID
337964
Card Set
Biochem LEC 1-3
Description
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Updated

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