Structure&Fuction of NT

  1. DNA Polymerase
    a. initially incorporates 1 wrong nucleotide sequence every ~106 bp

    b. overall accuracy is much higher at 1 error per 109 bp
  2. When an incorrect base pair is recognized, what actions take place?
    DNA polymerase reverses its direction by one base pair of DNA.

    • The 3’-5’ exonuclease activity of the enzyme allows the incorrect
    • base pair to be excised.

    Following this excision, the polymerase can re-insert the correct base and replication can continue.
  3. DNA replication
    • 1. bidirectional and semi-conservative
    • 2. Synthesis only in the 5’ to 3’ direction
    • 3. requires processive and distributive polymerases
    • 4. requires RNA primers on both strands near origin of replication5. highly dependent on Watson-Crick base paring
  4. What Proteins and enzymes are used in DNA replication?
    RNase H, DNA ligase, DNA polymerase δ, single-stranded DNA binding proteins (SSBs), topoisomerases, helicase, primase, replication protein A (RPA), FEN1
  5. DNA replication: Accuracy due to
    proofreading of polymerases and DNA repair system
  6. DNA Replication on lagging strand
    • 1. RNA primer hybridizes to ssDNA (synthesized by primase or RNA polymerase)
    • 2. elongation by a DNA polymerase, generating Okazaki fragments
    • 3. when the okazaki fragment encounters the next RNA primer, RNase activity (or 5’ to 3’ exonuclease activity by polymerase I in prokaryotes) removes the primer (additional FEN1 in eukaryotes)
    • 4. gap filling (by distributive polymerase)
    • 5. nick sealing (by DNA ligase
  7. Double-stranded DNA is fairly stable and strand separation usually requires high energy.
    1. Instead of high temperature, organisms use?
    2. They begin at the origin, which tends to be rich in
    • 1 Enzymes (ex: DNA helicase, DNA
    • Polymerase, RNase, DNA Topoisomerase) in order to bypass this problem.

    • 2 AT because they are easier to break
    • since they have only two hydrogen bonds whereas GC possesses three hydrogen bonds.
  8. Okazaki fragment x2
    1. short fragment of DNA created on the lagging strand during DNA replication

    2. synthesis is initiated from an RNA primer at the 5’ terminus
  9. Polymerase I
    DNA repair (gap filling); has 5'->3' activity and both 3'->5' exonuclease(Proofreading) and 5'->3' exonuclease activity (RNA Primer removal)
  10. Polymerase II
    involved in reparation of damaged DNA; has 3'->5' exonuclease activity
  11. Polymerase III
    main polymerase in bacteria (elongates in DNA replication); has 3'->5'exonuclease proofreading ability
  12. Exonucleases are enzymes that work by?
    cleaving nucleotides one at a time from the end of a polynucleotide chain. A hydrolyzing reaction occurs that breaks phosphodiester bonds at either the 3’ or 5’ ends.
  13. DNA ligase has applications in both?
    DNA repair and replication
  14. DNA ligase 2 functions ?
    1. links together two DNA strands that have double-strand breaks (a break in both complementary strands of DNA)

    2. fixes a single-stranded break where it uses the complementary strand as a template but requires the ligase to create the final phosphodiester bond for full repair.
  15. DNA Topoisomerases
    1. unwind/wind DNA to control the synthesis of proteins & DNA replication

    2. untwist the parental strand of DNA (transesterification).
  16. Topoiosomerase I
    transient breaks in one strand
  17. Topoisomerase II
    breaks in both strands

    a. essential in bacteria for replication and drug target for antibiotics

    b. essential in humans as a target for cancer chemotherapy
  18. E. coli & the Polymerases
    • 1. DNA polymerase I contains a 5' exonuclease that clips off the RNA primer contained immediately upstream from the site of DNA synthesis in a 5' → 3' manner
    • 2. the 5' -> 3' exonuclease activity makes it unsuitable for many applications
    • a. can be removed from the holoenzyme to leave a useful molecule (Klenow fragment)
  19. Processive vs. Distributive
    • 1. processive enzyme: used in the sense of going in a forward direction (5’->3’) and continues down this path without coming unbound, as in DNA Polymerase III.
    • Ex: polymerase that carries out many additions of nucleotide b4 dissociating from DNA
    • 2. distributive enzyme: catalyzes one cleavage first and then another one on another chain at random (gap filling), as in DNA Polymerase I.
  20. Telomere x3
    • 1. region of repetitive DNA at the end of a chromosome
    • 2. protects the end of the chromosome from deterioration
    • 3. “caps” the end of the chromosome, protecting it from homologous recombination and non-homologous end joining.
  21. Holliday junction
    • a. intermediate in homologous genetic recombination
    • b. important in maintaining genomic integrity
    • c. exists in prokaryotes and eukaryotes
  22. Heteroduplex
    • double-stranded (duplex) molecule of nucleic acid originated through the genetic recombination of single complementary strands derived from different sources, such as from different homologous chromosomes
    • Ex: Regions where one strand was from one duplex, and the other from the homologous duplex.
  23. Transposons
    • sequences of DNA that can move around to different positions
    • a. process called transposition catalyzed by transposases
    • b. two types: simple and replicative transposition
    • c. can cause mutations and change the amount of DNA in the genome
    • d. insertion sequences possess inverted repeats on both ends
    • e. they are flanked by direct repeats (same orientation) in the largest sequence
  24. demethylation: methylation of guanine bases is directly reversed by the protein
    methyl guanine methyl transferase (MGMT). The reaction is stoichiometric rather than catalytic
  25. DNA methylation pattern assists in distinguishing the parental strand from the newly synthesized strand, allowing for?
    mismatch repair if a wrong base is incorporated during replication
  26. base excision repair
    • cellular mechanism that repairs damaged DNA throughout the cell cycle. It is primarily responsible for removing small, non-helix distorting base lesions from the genome.
    • i. removal of damaged base by specific glycosidases
    • ii. repair of AP sites left after removal of a damaged base (or by spontaneous hydrlysis)
    • iii. AP endonucleases/AP lyases excise a basic sugar, gap filling, ligation
  27. nucleotide excision repair
    • prevent unwanted mutations by removing the vast majority of photodimers
    • i. recognize bulky distortions in the shape of the DNA double helix
    • ii. endonucleases excise a stretch of nucleotides
    • iii. gap-filling in by DNA polymerase I
    • iv. ligation by DNA ligase
    • v. mismatch: if wrong base is incorporated but is undamaged
    • vi. transcription-coupled: lesions block transcription but transcribed regions are repaired faster
  28. Both nucleotide excision repair and base excision repair systems require an
    endonuclease and a DNA ligase
  29. Translesion Synthesis
    a. damage tolerance process that allows the DNA replication machinery to replicate past DNA lesions such as thymine dimers or AP sitesb. involves switching out regular DNA polymerases for specialized translesion polymerases (e.g. DNA polymerase V),
  30. Recombination Repair
    a. daughter strand gap repair
    • i. piece of daughter strand replaces damaged portion of parental strand
    • ii. normal DNA duplex then repairs daughter strand
    • iii. normal DNA duplex replicates missing portion given to daughter strand
    • iv. most likely to occur on lagging strand
  31. Recombination Repair
    b. double-strand break repair
    • i. results because of ROS or topoisomerase inhibitors
    • ii. can be homologous (yeast) or nonhomologous end joining (mammalian cells)
  32. Recombination Repair
    c. repair of replication forks
    • i. recA binds to ssDNA regions at blocked replication forks to maintain integrity
    • ii. fork regression by branch migration (in protected state)

    • iii. lesion repair by base or nucleotide expansion repair
    • iv. reversal of branch migration
  33. Bypass Polymerases
    • a. induced upon extensive DNA damage (SOS reponse)
    • b. readily dissociates from DNA (distributive process so low processivity)
    • c. ensure continued replication if a lesion is encountered by the replication machinery
    • d. error prone and lack proofreading ability
    • e. induced to prevent a long halt of replication at lesions
  34. DNA bases

    A, T, G, C (in RNA, U replaces T)
  35. 1. Prokaryotes
    • a. 1 polymerase
    • b. specific σ factors recognize class of genes
    • ex) σ factor binds to RNA polymerase core enzyme
    • c. RNA polymerase binds to the promoter region first
  36. 2. Eukaryotes
    a. 3 polymerase

    b. transcription factors bind to DNA sequences

    c. transcription factors (or TATA binding protein (TBP)) binds to the promoter region first

    • d. require transcription factors but not σ factors for transcription
    • i. specific transcription factors recognize gene-specific DNA sequences
    • ii. transcription factors are proteins that oftentimes form dimers
  37. DNA is protected from alkylation (dimethyl sulfate) where enzyme is bound
    • i. Pribnow (AT-rich)
    • ii. -35 region (conserved and asymmetrical)
  38. certain nitrogens usually involved in H-bonding only gets alkylated if ssDNA
    i. “open” complex from middle of Pribnow box to around start site (-9 to +2)

    ii. easier for AT than GC (Pribnow box now AT-rich)

    iii. increase of GC pairs would decrease promoter efficiency

    iv. increase of AT pairs would INCREASE promoter efficiency
  39. Direction of Transcription
    • 1. RNA strand is read from 3’ to 5’
    • 2. RNA is synthesized (transcribed) in the 5’ to 3’ direction
  40. RNA vs. DNA synthesis
    1. RNA polymerase does NOT require a primer

    a. RNA polymerase molecules can transcribe one gene simultaneously

    2. DNA polymerase DOES require a primer
  41. Intercalating chemotherapies
    1. Daunomycin and Adriamycin

    2. inhibits initiation of RNA transcription
  42. Accuracy of RNA Transcription
    • 1. doesn’t have to be as accurate as that of DNA
    • a. genetic code degenerate (errors may be silent)
    • b. quick degradation and re-synthesis (high turnover rate)
    • c. one AA substitution in protein is oftentimes without consequence

    • 2. no repair system known
    • a. degradation and re-synthesis
    • b. high turnover rate
  43. RNA Processing

    1.Coupled Transcription-Translation
    for Prokaryotes and Eukaryotes
    • 1. Prokaryotes: occurs because prokaryotic mRNA does not need to be transported or processed, therefore, translation can begin immediately after transcription
    • 2. Eukaryotes: does not occur because mRNA must be processed and transported before translation can take place
  44. RNA Processing

    RNA modification for prokaryotes and eukaryotes
    1. Prokaryotes: protein translation often occurs with little or no modification

    2. Eukaryotes: extensive modification is possible
  45. Eukaryotic mRNA
    1. 5’-terminal cap (7-methyl G)

    2. 3’-terminal poly A tail

    3. methylated internal variants

    4. splice variants
  46. Splicing
    • 1. modification of RNA after transcription
    • 2. excision of intron sequences (noncoding) and exons are joined
    • 3. needed before eukaryotic mRNA can be used for translation
    • 4. involves several transesterification processes
    • 5. involves the formation of a 2’,5’-phosphodiester bond
    • 6. requires Watson-Crick base pairing with other RNA molecules for high accuracy
  47. RNA polymerase I
    RNA polymerase I carries out the transcription in eukaryotes3. in

    prokaryotes, transcription factors are not involved in rRNA transcription
  48. small interfering RNAs (siRNAs)
    • 1. they have a length of approximately 21-23 nucleotides
    • 2. they are double-stranded
    • 3. they inhibit gene expression by degrading homologous mRNA
    • 4. function (posttranscriptional) gene splicing (degradation of homologous mRNA sequences)
  49. What are three functions of salvage pathways?
    • Important drug targets for treatment of microbial diseases and/orparasitic diseases. Sites for manipulation of biological systems mutagenesis studies preparation of antibodies
    • Biological processes where genetic alterations have severe and far-reaching consequences
  50. What is the branch point in purine nucleotide synthesis?
  51. What is a difference between DNA and RNA polymerase with respect to the necessity for primers?
    DNA polymerase needs primers while RNA polymerase does not
  52. Consequences if DNA synthesis would occur from 3→5
    Top: energy for chain elongation would come from hydrolysis of triphosphate of chain

    Bottom: removal of a wrong paired 5’-terminal nucleotide triphosphate during proof reading would prevent further chain elongation
  53. DNA synthesis on lagging strand requires
    RNA fragments as primers (8-10 nt in humans; synthesized by primase or RNA polymerase) for synthesis of Okazaki fragments
  54. 1. RNA primer removal
    2.Gap filling
    3. Ligation of nick
    • RNase Hybridase
    • DNA Polyermase
    • DNA Ligase
  55. DNA Replication:
    Accessory proteins: Sliding clamps
    • Task: holding DNA polymerase in contact with DNA
    • Allows DNA polymerase to be more processive
    • (executes many reactions before dissociating
  56. 1 DNA polymerase III

    2 DNA polymerase I
    pol III, main, processive

    (pol I, gap filling, distributive) + 3’ → 5’ proofreading exonuclease activity + 5’ → 3’ exonuclease activity (RNase H; primer removal)
  57. Topoisomerase in humans and backteria
    In bacteria: Topoisomerase II is essential for replication and drug target for antibiotics

    In humans: Topoisomerase II is essential: target for cancer chemotherapy
  58. origins of replication (ori)

    replication bubbles move
    Eukaryotes (more DNA!) Thousands of origins (every 3-300 kb, tissue- and species-dependent)

    Prokaryotes one single ori

    • Termination requires topoisomerase II
    • bidirectionaly
  59. 1.DNA synthesis occurs during

    2.Drugs that inhibit DNA replication cause
    1. S phase of cell cycle

    2. G1- (longest phase of growth) or S cell cycle arrest
  60. Recombination
    Exchange of genetic info. can be homologous (identical sequence) or non homologous
  61. DNA Repair

    Q: How to recognize newly synthesized strand (and not "repair" parental strand)?
    A: Look at methylation pattern! Higher degree of methylation in parental strand
  62. DNA Repair
    Bypass polymerases are only induced upon
    extensive DNA damage (SOS response) and readily dissociates from DNA (distributive)
  63. DNA Repair

    Repair of Replication Forks
    Stop of replication on continuous strand would be detrimental (controlled by ori)
    • Putative mechanism:
    • 1. RecA binds to ssDNA regions at blocked replication forks to maintain integrity
    • 2. Fork regression by branch migration (in protected state)
    • 3. Lesion repair by base or nucleotide excision repair
    • 4. Reversal of branch migration
  64. RNA Transcription x3

    • 1.Initiation recognition of DNA sequence by RNA polymerase
    • 2.Elongation RNA chain synthesis
    • 3.Termination release of RNA chain/RNA polymerase from DNA template

    Prokaryotes: 1 polymerase + specific σ factors recognize classes of genes

    Eukaryotes: 3 polymerases + transcription factors bind to DNA sequences
  65. RNA Transcription

    How were promoter regions found? Footprinting
    1. DNA protected from alkylation (dimethyl sulfate) where enzyme is bound → Pribnow and -35 region

    2. Certain nitrogens usually involved in H-bonding only gets alkylated if ssDNA → "open" complex from middle of Pribnow box to ca. start site (-9 to +2)

    Easier for A:T than for G:C (Note: Pribnow box is AT rich)
  66. RNA polymerase
    Doesn not require primer
  67. RNA Transcription

    Tolerable precision
    Tolerable precision

    1 wrong base per 104 transcribed bases -genetic code degenerate -one aa substitution in protein often w/out consequences

    Genes can be transcribed by several RNA polymerase molecules at the same time
  68. Protein Translation =
    Ribosomal peptide synthesis from mRNA matrix
  69. pont mutation


    frameshift results from?
    1.Amino acid code unchanged

    2.Stop codon

    3.Change of amino acid code

  70. Ribosome have ____ tRNA binding sites
    three tRNA binding sites: A, P, E
  71. Protein Translation

    4 general steps



    4.Posttranslational Modification
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Structure&Fuction of NT