08 - DNA Replication & Repair

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Tookie
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280635
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08 - DNA Replication & Repair
Updated:
2014-08-22 20:16:23
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dna replication repair
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CMBM
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DNA Replication & Repair
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  1. To what end of the growing strand are the nucleotides added during DNA synthesis?
    3’ end
  2. In what direction do new DNA strands grow?
    5’ to 3’
  3. How is the new nucleotide added to the growing strand?
    dNTPs are substrates, and the α phosphate on the dNTP is retained on the strand with the β and γ phosphates being released as pyrophosphate; the hydrolysis of pyrophosphate to phosphate drives the rxn forward
  4. How do nucleoside analogs inhibit DNA replication?
    Most of the nucleoside analogs used as drugs lack a 3’-OH group, and therefore act as chain terminators
  5. How is the origin of replication defined?
    It is a specific DNA sequence which is AT rich; there is one in prokaryotes and several in eukaryotes
  6. What interactions direct DNA synthesis?
    Protein-DNA and protein-protein interactions
  7. What does helicase do?
    • Catalyzes unwinding of the DNA strand; it is unidirectional and energy-dependent; multiple helicases are present in prokaryotes &
    • eukaryotes
  8. How is the inherently unstable ssDNA kept stable?
    ssDNA-binding proteins bind ssDNA rightly and shift the equilibrium between ssDNA and dsDNA towards ssDNA; it prevents nucleases from attacking the ssDNA
  9. How is positive supercoiling dealt with during DNA replication as a result of unwinding the DNA strand?
    DNA topoisomerases introduce swivel points along the double helix; type1 makes single strand nicks and relaxes one supercoil at a time; type2 cuts both strand of DNA and relaxes two supercoils at a time; both topoisomers are ATP dependent
  10. How are topoisomerases drug targets?
    In anti-cancer drugs they are targeted to stop the process of replication, generating single or double strand breaks in the DNA; in anti-bacterial drugs they inhibit gyrase to stop replication & transcription, and generate double strand breaks in DNA
  11. What is the purpose of the RNA primer?
    • Since DNA pol cannot initiate the synthesis of a new strand, it can only catalyze the addition of a nucleotide to the 3’-OH group of a growing
    • strand, RNA primer provides the initial free 3’-OH group
  12. What is required for DNA synthetase activity?
    Primer, template, Mg2+/Zn2+ and all four dNTPs
  13. 3’ to 5’ exonuclease
    Proofreads the newly synthesized DNA strand; if an incorrect nucleotide is incorporated then DNA synthesis is paused b/c of incorrect positioning of 3’-OH; the last 2-4 bps unwind, and the unwound region of primer binds to the 3’ to 5’ exonuclease site; the incorrect nucleotide is removed, and the primer-template junction is reformed and rebind to the synthetase site
  14. 5’ to 3’ exonuclease
    Degrades RNA or DNA strands that are hybridized to template DNA strands; in prokaryotes this removes RNA primers
  15. Β-subunit of polymerase
    Present in enzymes with high processivity; it works as a sliding clamp, encircling the dsDNA to ensure that polymerase stays at the primer-template junction and is responsible for the processivity of the polymerase enzyme
  16. Pol I
    Found in prokaryotes; has low processivity, adding only ~50 nucleotides/min/enzyme molecule; it is mostly involved in RNA primer removal and DNA repair
  17. Pol II
    Found in prokaryotes; lacks proof reading activity, however is involved in DNA repair
  18. Pol III
    Found in prokaryotes; has high processivity, adding ~9000 nucleotides/min/enzyme molecule; replicates most of the DNA
  19. Pol IV & Pol V
    Found in prokaryotes; involved in DNA repair
  20. Replisome
    Complex, found in prokaryotes, comprised of a helicase, ssDNA-binding proteins, primase and dimeric Pol III holoenzyme; this catalyzes the synthesis of both strands of DNA
  21. What are the final steps in DNA replication in prokaryotes?
    The removal of the RNA primer is mediated by DNA Pol I, and 5’ to 3’ exonuclease removes NTPs from the 5’ end; the synthetase activity adds dNTPs to the 3’ end of the previous strand; the okazaki fragments are sealed by DNA ligase and 2 circular DNA molecules linked together in a chain is untangled by topoisomerase
  22. Pol α
    Found in eukaryotes; tightly interacts with primase
  23. Pol β
    Found in eukaryotes; functions in repair
  24. Pol γ
    Found in eukaryotes; mitochondrial polymerase
  25. Pol δ
    Found in eukaryotes; polymerase of the lagging strand; has proofreading activity (3’ to 5’ exonuclease activity)
  26. Pol ε
    Found in eukaryotes; polymerase of the leading strand; has proofreading (3’ to 5’ exonuclease) activity
  27. RNaseH
    Removes RNA primers in eukaryotic cells
  28. Polymerase switching
    Replication is initiated by Pol α/primase comples; after Pol α incorporates a few nucleotides, Pol δ or Pol ε replaces Pol α
  29. What are endogenous sources of DNA?
    Replication errors; spontaneous alteration in the chemistry of DNA bases [cytosines deaminate to uracil, and 5-metylcytosines deaminate to thymine (these are hotspots for mutations)]; transposons
  30. Alkylating agents
    Source of DNA damage; alkyl groups are covalently attached to bases
  31. Crosslinking Agents
    Source of DNA damage; inter-strand crosslinking by covalent linkage
  32. Base analogs
    Source of DNA damage; substitute for normal bases
  33. Intercalators
    Source of DNA damage; intercalate between bps and cause insertion or deletion mutations
  34. What is the general mechanism of DNA repair?
    Proteins recognize deformity in DNA molecule; recruits endonuclease and other pathway-specific enzymes; exonucleases fix problem
  35. Direct reversal of DNA damage
    Involved in the removal of alkyl groups attached to the O6 in guanine; mediated by O6-methylguanine methyl transferase (MGMT) or alylguanine alkyl transferase (AGAT)
  36. Mismatch repair (MMR)
    • Repair of replication errors and small insertion or deletion; Mut proteins are involved in the
    • recognition of the mismatch, make a nick in the newly synthesized strand; defects in the MMR pathway can lead to sporadic cancers
  37. Lynch Syndrome
    Previously called hereditary non-polyposis colon cancer (HNPCC) commonly has mutation in the Mut protein homologs
  38. Base excision repair (BER)
    Repair of damaged bases or ssDNA breaks; generally works by removal of the base by a glycosylase, cleaving the sugar-phosphate backbone by an AP endonuclease which removes the abasic nucleotide, insertion of the correct nucleotide by polymerase, and ligation of the backbone by ligase
  39. Nucleotide excision repair (NER)
    Repairs a variety of helix-distorting DNA lesions such as pyrimidine dimer, bulky adduct of base, cisplatin-induced DNA damage; after defect recognition, nucleotides are removed by pathway-specific exonuclease and helicase and the gap is filled in by DNA polymerase; also called the global NER pathway because it scans the entire genome for mutations
  40. Xeroderma Pigmentosum
    Results for a defect in the global NER pathway; first sign is usually excessive, long-lasting sunburn, dry skin, excessive freckling, eye damage, and keratosis; has a 2000-fold higher rate of fatal skin cancer
  41. Cockayne’s syndrome
    Results from defect involved in transcription-coupled NER pathway; RNA synthesis following exposure to UV is drastically reduced; results in dwarfism and developmental delay
  42. Homologous recombination pathway
    HR repairs the dsDNA break by using a duplicate set of DNA as the template; this is a very precise pathway; this is particularly active during the late S or early G2 phase of the cell cycle; BRCA1 and BRCA2 are involved in the stabilization of the breaks and promoting HR over NHEJ
  43. Nonhomologous end joining
    NHEJ repairs the dsDNA break directly w/o any outside information; the proteins involved in the pathway bind the free ends of the broken DNA, trim the ends and ligate them back together; this may result in the addition or removal of a few nucleotides at the repair site; used to repair most of the double strand breaks, particularly during the G1 phase

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