Chapter 11 DNA replication and recombination

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  1. Three types of DNA replication
    • conservative
    • semiconservative
    • dispersive
  2. Conservative
    original helix is conserved and two newly synthesized strands come together
  3. Semiconservative
    each replicated DNA molecule consists of one "old"strand and one new strand
  4. Dispersive
    Parental strands are dispersed into two new double helices
  5. Messelson and Stahl
    used 15N and 14 N to demonstrate that DNA replication is semiconservative
  6. Taylor-woods-hughes
    • using broad bean vicia faba
    • demonstrated that DNA replication is semiconservative in eukaryotes
  7. Replication fork
    • created when replication is occuring
    • strands of helix are unwound
    • Bidirectional; 2 replication forks at one origin site of replication
  8. Replicon
    length of DNA that is replicated following one initiation event at a single origin
  9. OriC
    origin of replication in which bacteria have a single circular DNA and the DNA synthesis originates at this single point
  10. DNA polymerase
    catalyzes DNA synthesis and requires a DNA template and all four deoxyribonucleoside triphosphates (dNTPs)
  11. How does chain elongation occur?
    • a nucleotide is added at the 3' end
    • as the nucleotide is added the two terminal phosphates are cleaved off and a newly exposed 3'-OH group can participate in the addition of another nucleotide as DNA synthesis proceeds
  12. DNA polymerase III
    enzyme responsible for the 5' to 3' polymerization essential in vivo
  13. Holoenzyme
    DNA polymerase III is a complex enzyme that is made up of 10 subunits
  14. Replisome
    • complex formed at the replication fork
    • consists of holoenzyme and some other proteins
  15. 7 key issues that must be resolved during DNA replication
    • 1) unwinding of the helix
    • 2) reducing increased coiling generated during unwinding
    • 3) synthesis of a primer for initiation
    • 4)discontinuous synthesis of the second strand
    • 5) removal of the RNA primers
    • 6) joining of the gap-filling DNA to the adjacent strand
    • 7) proofreading
  16. DnaA
    binds to the origin of replication and is responsible for the initial steps in unwinding the helix
  17. DnaB and DnaC
    further open and destabilizes the helix when binded
  18. Helicases
    proteins which require the energy normally supplied by the hydrolysis of ATP to break hydrogen bonds and denature the double helix
  19. Single-stranded binding proteins (SSBPs)
    stabilize the open conformation
  20. Supercoiling
    produced from unwinding
  21. DNA gyrase
    • relieves supercoiling
    • belongs to a larger group of enzymes referredt o as DNA topoisomerases
    • Gyrase makes single or double stranded cuts to undo the twists and knots created during supercoiling which are then resealed
  22. Primase
    • synthesizes an RNA primer that provides the free 3-hydroxyl required by DNA polymerase III
    • initiates DNA synthesis
  23. DNA polymerase I
    removes primer and replaced with DNA
  24. Leading Strand
    continuous DNA synthesis; the main template
  25. Lagging starnd
    • undergoes discontinuous DNA synthesis
    • synthesized as Okazaki fragments, each wtih an RNA primer
  26. DNA ligase
    joins Okazaki fragments after primers are removed by DNA polymerase I
  27. B-subunit clamp
    prevents the core enzyme from falling off the template during DNA synthesis
  28. Proofreading and error correction
    • integral part of DNA replication
    • All of the DNA polymerases have a 3' to 5' exonuclease activity that allows proofreading
  29. DNA synthesis at a single replication fork involves:
    DNA polymerase III, single-stranded binding proteins, DNA gyrase, DNA helicase, and RNA primers
  30. Conditional mutations
    a number of genes involved in DNA replication
  31. Temperature-sensitive mutation
    • example of a conditional mutation
    • may not be expressed at a particular permissive temperature but when mutant cells are grown at a restrictive temperature, the mutant phenotype is expressed and can be studied
  32. Differences in eukaryotic DNA replication and prokaryotic DNA
    • eukaryotes have more DNA than prokaryotes
    • chromosomes are linear
    • DNA is complexed with proteins
    • eukaryotes contain multiple origins of replication to allow the genome to be replicated in a matter of mins to a few hours
  33. Autonomously replicating sequences (ARSs)
    • yeast genomes contain 250-400 origins
    • these sequences contain an 11-bp consensus sequence flanked by other short sequences involved in efficient initiation
  34. Prereplication complex (pre-Rc)
    assembles at replication origins
  35. Origin recognition complex (ORC)
    • in early G1 phase of cell cycle
    • replication origins are recognized by a six-protein complex
    • tags the origin as the site of initiation
  36. How many DNA polymerases are involved in replication of mitochondrial DNA?
  37. Polymerase alpha, delta, and epsilon
    major forms of enzyme involved in initiation and elongation
  38. Processivitiy
    term that reflects the length of DNA that is synthesized by an enzyme before it dissociates from the template
  39. Polymerase alpha
    • possesses low processivity
    • synthesis of RNA primers during initiation on the leading and lagging strands
  40. Polymerase switching
    once primer is in place, polymerase alpha and epsilon are replaced by polymerase delta for elongation
  41. Polymerase Delta
    synthesizes lagging strand
  42. Polymerase Epsilon
    synthesizes lagging strand
  43. Telomeres
    ends of linear chromosomes consists of long stretches of repeating sequences and preserve the integrity and stability of chromosomes
  44. Telomerase
    • directs synthesis of the telomere repeat sequence to fill the gap
    • this is an enzyme consisting of a ribonucleoprotein with an RNA that serves as the template for the synthesis of its DNA complement (reverse transcription)
  45. Reverse transcription
    DNA is made form an RNA template
  46. Malignant cells and telomerase
    malignant cells maintain telomerase activity and are immortalized
  47. Genetic recombination involves
    • endonuclease nicking
    • strand displacement and pairing with complement
    • ligation
    • branch migration
    • duplex separation to generate the characteristic Holliday structure
  48. General, homologous, recombination
    genetic exchange at equivalent positions along two chromosomes with substantial DNA sequence homology
  49. RecA
    protein in E. coli promotes the exchange of reciprocal single-stranded DNA molecules and enhances hydrogen bond formation during strand displacement
  50. Gene conversion
    characterized by nonreciprocal genetic exchange between two closely linked genes
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Chapter 11 DNA replication and recombination
2013-10-28 19:52:32

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