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DNA Replication

DNA Replication. Chapter 25. DNA Polymerase (E. coli ex). Catalyzes synth of new DNA strand d(NMP) n + dNTP  d(NMP) n+1 + PPi 3’ –OH of newly synth’d strand attacks first phosphate of incoming dNTP Rxn thermodynamically favorable Why??. DNA Polymerase – cont’d.

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DNA Replication

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  1. DNA Replication Chapter 25

  2. DNA Polymerase (E. coli ex) • Catalyzes synth of new DNA strand • d(NMP)n + dNTP  d(NMP)n+1 + PPi • 3’ –OH of newly synth’d strand attacks first phosphate of incoming dNTP • Rxn thermodynamically favorable • Why??

  3. DNA Polymerase – cont’d • Noncovalent stabilizing forces impt • REMEMBER?? • Base stacking  hydrophobic interactions • Base pairing  multiple H-bonds between duplex strands • As length of helix incr’d, # of these forces incr’d incr’d stabilization

  4. DNA Polymerase – cont’d • Can only add nucleotides to pre-existing strand • So problematic at beginning of repl’n • Problem solved by synth of ….

  5. DNA Polymerase – cont’d • Primers (25-5) • Synth’d by specialized enzymes • Nucleic acid segments complementary to template • Often RNA • Have free 3’ –OH that can attack dNTP

  6. DNA Polymerase – cont’d • Once DNA polymerase begins synth of DNA chain, can dissociate OR can continue along template adding more nucleotides to growing chain • Rate of synth DNA depends on ability of enz to continue w/out falling off • Processivity

  7. DNA Polymerase – Accuracy • Enz must ACCURATELY add correct nucleotide to growing chain • E. coli accuracy ~ 1 mistake per 109 – 1010 nucleotides added • Geometry of enz active site matches geom. of correct base pairs (25-6) • A=T, G=C fit • Other pairings don’t fit

  8. Fig.25-6

  9. Accuracy – cont’d • Enz has “back-up” proofreading ability • Its conform’n allows recognition of improper pairing • Has ability to cleave improperly paired bases (25-7) • Called 3’  5’ exonuclease activity • Enz won’t proceed to next base if previous base improper • Then catalyzes add’n of proper base • Increases accuracy of polymerization 102 – 103 X • Note: cell has other enz’s/mech’s to find/repair mistakes (mutations) after new helix synth’d w/ repl’n

  10. Fig.25-7

  11. 3 E. coli DNA Polymerases (Table 25-1) • I -- impt to polymerase activity • Slow (adds 16-20 nucleotides/sec) • Has 2 proofreading functions • Only 1 subunit • II – impt to DNA repair • Less polymerization activity • Several subunits

  12. 3 DNA Polymerases -- cont’d • III – principle repl’n enz • Much faster than polymerase I (adds 250-1000 nucleotides/sec) • Many subunits (Table 25-2) each w/ partic function • Encircles DNA; slides along helix (25-10) • One subunit “clamps” helix  better processivity

  13. Fig.25-10

  14. Replisome • Many other enz’s/prot’s necessary for repl’n (Table 25-3) • Complex together  replisome • Helicases – sep strands • Topoisomerases – relieve strain w/ sep’n • Binding proteins – keep parent strands from reannealing • Primases – synth primers • Ligases – seal backbone • What bonds hold nucleic acid backbone together?

  15. Initiation – 1st Stage Repl’n • E. coli unique site = ori C (25-11) • 3 adjoining 13-nucleotide consensus seq’s • Non-consensus “spacer” nucleotides • 4 9-nucleotide consensus seq’s spaced apart • Consensus seq’s contain nucleotides in partic seq common to many species

  16. Initiation – cont’d • At ori C (at 4 9-‘tide seq area) (25-12) • ~20 DnaA mol’s (proteins) bind • Requires ATP •  nucleosome-like structure

  17. Initiation – cont’d •  Unwinding of helix (at 3 13-‘tide seq area) • ~13 nucleotides participate in unwinding • Requires ATP • Requires HU (histone-like protein)

  18. Initiation – cont’d • Unwound helix is stabilized • Requires DnaB, DnaC (proteins) • These bind to open helix • DnaB also acts as helicase • Unwinds DNA helix by 1000’s of bp’s

  19. Initiation – cont’d Result: • Nucleotide bases now exposed for base pairing in semiconservative repl'n • What does semiconservative mean? • Yields 2 repl’n forks

  20. Initiation – cont’d • Other impt repl’n factors at repl’n forks (Table 25-4) • SSB = Single Strand DNA Binding Protein • Stabilizes sep’d DNA strands • Prevents renaturation • DNA gyrase -- a topoisomerase • Relieves physical stress of unwinding • Note: in E. coli, repl’n is regulated ONLY @ initiation

  21. Elongation • Second stage of repl’n • Must synth both leading and lagging strands • REMEMBER: 1 parent strand 3'  5; its daughter can be synth'd 5'  3' easily. What about the other parent strand (runs 5'  3')?? • Follows init’n w/ successful unwinding  repl’n fork, stabilized by prot’s • So have parent strands available as templates for base-pairing  2 daughter dbl helices

  22. Elongation -- cont'd – Leading Strand • Simpler, more direct (25-13) • Primase (=DnaG) synthesizes primer • 10-60 nucleotides • NOTdeoxynucleotides • Short RNA segment • Occurs @ fork opening • Yields free 3’ –OH that will attack further dNTP’s

  23. Leading Strand – cont’d • DNA polymerase III now associates • Catalyzes add’n of deoxy-nucleotides to 3’ –OH (25-5)

  24. Leading Strand – cont’d • Elongation of leading strand keeps up w/ unwinding of DNA @ repl’n fork • Gyrase/helicase unwind more DNA  further repl’n fork • SSB stabilizes single strand DNA til polymerase arrives • Synth continues 5’  3’ along daughter strand

  25. Fig.25-13

  26. Elongation -- cont'd – Lagging Strand • More complicated • REMEMBER: still need 5’  3’ synth, AND still need to have antiparallel strands. • Template strand here is 5’  3’ • Can’t synth continuous daughter strand 5’  3’ • Cell synth’s discontinuous DNA fragments (Okazaki fragments) that will be joined (25-13) • Must have several primers AND coordinated fork movement

  27. Fig.25-13

  28. Lagging Strand – cont’d • Lagging strand is looped next to leading strand (25-14) • DNA polymerase III complex of subunits catalyzes nucleic acid elongation on both strands simultaneously • Primosome = DnaB, DnaG (primase) held together w/ DNA polymerase III by other prot’s

  29. Fig.25-14

  30. Lagging Strand – cont’d • One subunit complex of DNA polymerase III moves along lagging strand @ fork in 3’  5’ direction (along parent) • Another subunit complex of polymerase III synth’s daughter strand along leading strand • At intervals, primase attaches to DnaB (helicase) • Here, primase catalyzes synth of primer (as on leading strand) • Also (once primer synth’d), primase directs “clamp” subunit of polymerase III to this site • This directs other polymerase III subunits to primer

  31. Lagging Strand – cont’d Now polymerase III catalytic subunits add deoxynucleotides to primer  Okazaki fragment • Book notes primosome moves 3’  5’ along daughter strand, but both primase & polymerase synthesize strands 5’  3’ along daughter

  32. Fig.25-14

  33. Lagging Strand – cont’d • Okazaki fragments must be joined • DNA polymerase I exonuclease cleaves RNA primer (25-15) • DNA polymerase I simultaneously synth’s deoxynucleotide fragment • 10-60 nucleotides • Nicks between fragments

  34. Lagging Strand – cont’d • DNA ligase seals nicks between fragments (25-16) • Catalyzes synth of phosphodiester bond • NADH impt (coordination role?)

  35. Fig.25-16

  36. Termination • Repl'n has occurred bidirectionally @ 2 forks concurrently • E. coli genome is closed circular • So 2 repl'n forks will meet

  37. Termination – cont’d • Ter = seq of ~ 20 nucleotides (25-17) • Tus = prot's that bind Ter • When replisome encounters Ter-Tus • Replisome halted • Repl'n halted • Replisome complex dissociates

  38. Termination – cont’d • Result = 2 intertwined (catenated) circles • Topoisomerase IV nicks chains • One chain winds through other • 2 Complete genomes sep'd

  39. Eukaryotic DNA Replication • Repl'n mechanism & replisome structures similar to prokaryotes, BUT: • DNA more complex • Not all is coding for peptides • Chromatin packaging more complex • REMEMBER: nucleosomes, 30 nm fibers, nuclear scaffold, etc. • No single origination pt for repl'n • Many forks develop •  Simultaneous repl'ns bidirectionally • Forks move more slowly than in E. coli • But efficient because more forks

  40. Eukaryotic DNA Replication • Repl'n enzymes not yet fully understood • DNA polymerase a • In nucleus • Has subunit w/ primase activity • May be impt to lagging strand synth • DNA polymerase d • Assoc'd w/ a • Impt to attaching enz to nucleic acid chain • Has 3'  5' exonuclease ability (proofreading) • DNA polymerase e • Impt in repair

  41. Eukaryotic DNA Replication • Replisome proteins not yet fully understood • Found prot's similar to SSB prot's of E. coli • Termination seems to involve telomerases • Telomeres = seq's @ ends of chromosomes

  42. DNA Alterations • Need unaltered, correct nucleotide seq to code for correct aa's  correct peptides  correct proteins • Some changes acceptable • Some "wobble" in genetic code • Some DNA damage in mature cells can be fixed • DNA repair mechanisms avail for TT dimers (ex) • Have (more) other mature cells that can maintain homeostasis in organism • BUT -- if mispaired bases during repl'n  mutation in daughter cell (and her subsequent daughters)

  43. Definitions • Lesion = unrepaired DNA damage • Mammalian cell prod's > 104 lesions/day • Mutation = permanent change in nucleotide seq • Can be replicated during cell division • Results if DNA polymerase proofreading fails • May occur in unimpt region = Silent Mutation • Doesn't effect health of organism

  44. Definitions – cont’d • Mutation -- cont’d • May confer advantage to organism = Favorable • Rare • Impt in evolution • May be catastrophic to organism health • Correlations between mutations & carcinogenesis

  45. DNA Repair • Cell has biochem mech's to repair damage to DNA • Though 104 lesions/day, mutations < 1/1,000 bp's • If repair mech's defective  disease/dysfunction • Ex: xeroderma pigmentosum • UV light  DNA lesions • No repair mech •  Skin cancers • Repair mech ex: base excision repair • Takes advantage of complementarity of strands

  46. Base Excision Repair • N-Glycosylases • Cleave N-glycosyl bonds • What parts of nucleic acid are joined by N-glycosyl bonds? • Several specific N-glycosylases • Each recognizes a common DNA lesion • Common -- bases altered by deamination events • Yields apurinic or apyrimidinic (AP) site

  47. Excision Repair – cont’d • Uracil Glycosylase -- ex • Deamination of cytosine  uracil (improper) • Enz recognizes, cleaves ONLY U in DNA • Not U in RNA • Not T in DNA •  AP site on DNA (25-22) • Would this be apurinic or apyrimidinic? • Leaves behind sugar-phosphate of original nucleotide

  48. Excision Repair – cont’d • Then other enz's (AP endonucleases) cleave several bases of mutated strand around AP site • Then DNA polymerase I catalyzes polymerization of proper nucleotides at site • Then DNA ligase seals nicks on sugar-phosphate backbone

  49. Fig.25-22

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