BIOL 200

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BIOL 200
2012-09-22 23:27:10

5th lecture
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  1. In what mechanism does DNA proceed in?
    Semiconservative mechanism 
  2. Nucleotide Polymerization 
    • Catalyzed by DNA polymerase 
    • requires deoxynucleotides 5'-triphosphates (dNTPs, where N= A,T,C or G)
    • requires a primer ( can be DNA or RNA)
    • proceeds from a 5' to 3' direction 
  3. How are free dNTPs added?
    Free dNTPs are added through the formationof a new phosphodiester bond involving the3’ hydroxyl of the terminal sugar.
  4. What happens if the primer is RNA in Nucleotide Polymerization
    If the primer is RNA then the addition ofdNTPs by DNA polymerase will result in amolecule that is RNA at the 5’ end andDNA at the 3’ end.
  5. Origins of Replication
    In order for duplex DNA to be replicated the strands must be separated. This is done through the participation of DNA helicases. Unwinding is initiated at aspecific region(s) in duplex DNA. Theseregions are referred to as the origins of replication (or origins).origins tend to be AT-rich
  6. Primer and Primase
    A specialized RNA polymerase, called a primase, forms a short RNA molecule complementary to a single-stranded region of the unwound duplex DNA. DNApolymerase extends the primer to eventuallyform a new daughter duplex DNA.
  7. Topoisomerase 
    Relieves supercoils
  8. Leading strand
    Happends in a continuous manner
  9. Lagging strand
    • Happens in a non continuous manner
    • primase lays down primers at different intervals 
  10. Okazaki segments
    Are RNA segments on the 5' end with DNA projections on the 3' end

    The RNA component ofthe Okazaki fragmentsare replaced by DNA andthe two adjoining DNAmolecules are ligatedtogether by DNA ligase.
  11. What makes the phosphodiester linkage
    DNA lligase
  12. Large T-antigen
    • Helicase from SV40 virus
    • Encoded by the viral genome 
    • hexamer 
  13. (replication protein A)RPA 
    • binds to single stranded DNA
    • Keeps the single stranded template DNA in optical confirmation for the DNA polymerase 
  14. What do DNA polymerase δ (Pol δ)Replication factor C (Rfc)Proliferating cell nuclearantigen (PCNA) do?
    • carry out leading-strand DNA synthesis
    • RPA is displaced assynthesis proceeds
  15. PCNA
    • Is a homotrimeric protein
    • Prevents the Pol δ/Rfc/PCNA complexfrom disassociating from the template.
  16. What do Primase/Pol α Complex do?
    Primase forms the RNAcomponent of the primer and DNA polymerase α (Pol α) extends the primer with DNA.
  17. What does Pol δ/Rfc/PCNA complex do?
    Replaces the Pol α/primase complex and completes the synthesis of an Okazaki fragment.
  18. Whats does Ribonuclease H and FEN-1 and Pol δ do?
    Ribonuclease H and FEN-1 displace the RNA component at the 5’ ends of the Okazaki fragments.

    Pol δ replaces the RNAwith DNA.

    DNA fragments are then ligated together by DNA ligase.
  19. DNA replication proceeds...
  20. Loading of Bidirectional DNA Replication
    • Helicase unwinding 
    • Leading-strand primer synthesis
  21. Unwinding Bidirectional DNA Replication
    Unwinding by large T-antigen(helicase) driven by the hydrolysis of ATP. RPAbinds to single stranded regions
  22. Primer Synthesis Bidirectional DNA Replication
    Primase-Pol α complexes synthesize primer sequences that are base-paired to the parental templates.
  23. Extension Bidirectional DNA Replication
    • Pol δ/Rfc/PCNA complexes replace the primase-Pol α complexes and extend the primer sequences.
  24. Further Unwinding Bidirectional DNA Replication
    Furtherunwinding andbinding of RPA tosingle-strandedregions.
  25. Further Extension Bidirectional DNA Replication
    • Pol δ/Rfc/PCNA complexes continue to synthesize the leading-strands.
  26. Lagging-strand Primer in Bidirectional DNA Replication
    Primase-Pol α complexes form primers for lagging-strand synthesis.
  27. Primer Extension, Primer Removal,and Strand Ligation in Bidirectional DNA Replication
    • Pol δ/Rfc/PCNA complexes replace the primase-Pol α complexes and extend the primer sequence.
    • Removal of the primers is performed by FEN-1 and ribonuclease H
    • Pol δ/Rfc/PCNA complexes replace the primer sequences with DNA
    • Strands are ligated together by DNA ligase.
  28. Eukaryotic chromosomal DNA contains multiple replication origins
    • Origin Recognition Complex (ORC)
    • six subunit protein
    • binds to origins
    • associates with other proteins to load helicases (MCM or minichromosome maintenance)