Ch 5 Notes

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  1. What are problems in replicating DNA that must be overcome?
    • 1) maitain downstream structure
    • 2) avoid supercoiling
    • 3) antiparallel
    • 4) fill in gaps
    • 5) H bonds-- melt and reform
    • 6) mistakes
    • 7) speed
  2. What was done to determine that OF were made on the allging strand?
    Use H3-thymidine pulse-chase eperiment

    DNA in a cell free system with building blocks, including radioactive thymidine, and all enzymes. 

    Start the process. Let the replication. Then, add a quick pulse where labeled thymidine is exposed. Let it go for a period of time and then flood the system with cold T to get rid of it called the chase
  3. pulse chase experiemt
    done with things we can't see well

    begins with no exposure to radioactive label. Then, in a pulse period, it exposes it to the radioactive label

    The chase is the period whre there is no more use of hte radioactive adn the nonradioactive is used

    Used to determine OF and the replication fork
  4. What are the three requirements for DNA polymerase?
    single strand DNA template strand

    building blocks: dNTPs

    free 3' OH end; DNA pol can't start on own
  5. Helicase structure
    six subunits: each subunit hydrolyzes ATP
  6. What drives the addition of nucleotides?
    the energy form ATP hydrolysis and the triphosphate allows the reaction to proceed
  7. DNA primase
    • DNA dependent RNA polymerase
    • depends on DNA template to synthesize a complemnetary RNA strand; capable of starting from scratch of a short stretch of RNA
  8. Explain nick sealing.
    DNA ligase fixes teh nick. It links together strands of DNA using energy through ATP

    DNA ligase finds broken region, grabs ATP, separates phosphates and repairs nick by fusing ATP to 5' end
  9. clamp loader
    responsible for the assembly of the clamp around DNA and providing the energy; five subunits; tightens around the primer junction and hydrolyzes ATP and loads the clamp; dissociates onece the clamp has been assembled
  10. sliding clamp
    keeps polymerase firmly on the DNA when it is moving, but releases it as soon as the polymerase runs into a double-stranded region of DNA
  11. What are the sites of DNA Pol?
    A polymerizing site and an editing site (exonucleolytic)

    In the editing mode, the newly synthesized DNA transiently unpairs from the template and the polymerase undergoes a conformational change, moving the editing catalytic site into place to remove the most recently added nucleotide
  12. What can repair enzymes detect?
    they scan along looking for bumps, incorporated as a mistake in the nucleotide sequence

    proteins latch on to get rid of strand part containing the bump. A nick is formed adn filled in with nucleotides
  13. Why is it in teh 5-->3 and not 3-->5?
    It allows the chain to continue to be elongated when a mistake in polymerizaton has been removed by exonucleolytic proofreading. 

    In the three to five direction, there would be blockage of further chain elongation. No, high energy bond would be cleaved, as opposed to if it were 5-->3, where a high energy bond can be cleaved
  14. plasmids
    usd to transfer DNA from one organism to another
  15. What was the experiment done to determine origins?
    Buffer put over DNA with enzymes to begin the process

    After a few minutes, put H3-thymidine and pulse for ten minutes. 

    Wash away in the chase to reduce the levels of radioactive thymidine

    demonstrated that the replication was moving away from the bubble in both directions, tapering off the farther along it went
  16. What is unique about the nucleotide sequnece at the replication origin?
    it contains sequence that attracts initiator proteins, which attract helicase in an inactive form (protein bound to it)

    An intermediate causes the inhibitor to be released and activated. Primase is then called. 

    Initiator binds to replication origin. Helicase in inactive form binds to initiator protien. Helicase loaded onto strand and activated, causing opening of the helix and binding of primase to form a primosome. DNA primase lays down primer. DNA pol starts the chain
  17. What was the experiment done with yeast cells?
    Colonies of yeast without ability to make histidine loaded on medium lacking histidine. 

    Genome cut into pieces and introduced into plasmids genetically modified with histdine. Plasmid put in yeast cells so it can make its own histidine in the plasmids are the fragments of the genome. 

    One fragment will have the origin of replication and cells with plasmid without origin don't pass histidine on
  18. What is inclued in the origin of replicaiton?
    there is an ORC binding site, where the origin replication complex will bind

    unwinding region, a region containing A-T sequences that can be easily unbound

    Abf1-binding site, where an auxilary protein, Abf, facilitates ORC binding (helper proteins help recruit ORC)
  19. histone chaperones
    ex: NAP-1 and CAF-1 restore the full complement of histones to daughter molecules
  20. telomerase
    RNA-dependent DNA polymerase: template being used is an RNA molecule that is bound to the protein; acts as a base-pairing enzyme
  21. What can occur on a daily basis?
    • depurination: N-glycosyl linkages to deoxyribose hydrolyze 
    • deamination: cytosol converts to uracil
  22. What effect can deamination have?
    one daughter strand will inherit a point mutaton
  23. What effect can depurination have?
    a deletion can occur in a daughter strand
  24. glycosylase
    functions in base excision repair to remove the base using a flipping out method; hydrolyzes the base from the sugar and removes it
  25. AP endonuclease
    along with phosphodiesterase removes the sugar phosphate
  26. Nucleotide excision repairs involves what?
    latching on of the enzyme upstream and dounstream and nicks the backbone on either side

    a helicase removes the damaged region

    larger gap: polymerase latches on to synthesize new strand
  27. What are the two types of homologous recombination?
    homologous: most frequently occurs in newly replicated DNA because sister chromatids still associated with each other; damage to one allows the other to fix it; the info exists on sister chromatids; used to fill in gap

    non-homologous: chromosome by itself that somehow a ds break has occurred; repair system finds it but results in loss of info; rescuing a damaged chromosome; if nick in both strands, blunt ends are created and joined--> loss of information
  28. Explain non-homologous recombination.
    Ku proteins (heterodimers that grasph broken chromosome ends) scan the genome and look for ds breaks. 

    Binds to the end of the break and recruits additional proteins, which hold the broken ends together while they are processed and joined covalently

    The checkpoint can't be passed until all fo the damage is fixed.
  29. Explain homologous recombination.
    When a moving replication fork encounters a single-strand break, it will collapse but can be repaired by homologous recombination. 

    The initial strand invasion requires a free 3' end generated by a nuclease that degrades the 5' end of the complementary strand. Recombination then begins with starnd invasion in which the strand being elongated uses the sister chromatid strand, whih is undamaged. Strand breakage then occurs, along with additional DNA synthesis. The replicaton fork then restarts
  30. How to hybridize strands.
    DNA double helices reform from their separated strands in a reaction that depends on the random collision of two complementary DNA strands.
  31. What happens in homologous DNA repair?
    RecA holds a double strand and a single strand. First, a non-base-paired complex is formed, which is converted into a joint molecule as a homologous sequence is found. 

    The RecA positions the strand so that it invades the other.
  32. Explain branching.
    Can be either spontaneous branch migration or protein-directed branch migratio. 

    In spontaneous branch migration, there is a random-walk process and makes little progress

    Protein directed requires ATP and moves the branch point at a uniform rate in one direction
  33. Holliday junction
    region where we have crossing over of chromatin; if rotation, you get recombinant DNA

    the two DNA strands switch partners between two double helices
  34. resolution
    cutting of the strands in Holliday junction
  35. Explain homologous recombination in meiosis.
    Rather than a randomly occurring break, there is a purposeful ds break. 

    One chromosome is cut and an exonuclease exposes the single stranded 3' end. Strand invasion joins the chromosomes, creating a double Hilliday junciton. 

    Two things can occur. The chromosomes can be cut directly, leading to a single region of crossover. Or, they can cut in different places, leading to several crossover regions
Card Set:
Ch 5 Notes
2015-02-05 02:40:38
Test One
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