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Part Two of BMB 400

Part Two of BMB 400. Enzymes needed for DNA replication: Chapter 5 Classes Sept 23, 25, Oct 02, 07 Covered entire chapter Origins, terminators and control of replication: Chapter 6 Classes Oct 04, 07. Note that supercoiling is from Chapter 2, pages 77-80 (questions 2.12-2.14)

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Part Two of BMB 400

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  1. Part Two of BMB 400 Enzymes needed for DNA replication: Chapter 5 Classes Sept 23, 25, Oct 02, 07 Covered entire chapter Origins, terminators and control of replication: Chapter 6 Classes Oct 04, 07. Note that supercoiling is from Chapter 2, pages 77-80 (questions 2.12-2.14) Cover all but “Cellular control of replication” pages 322-326, question 6.11, 6.18, 6.19 Mutation and Repair: Chapter 7 Class: Oct 09 Restrict coverage of mutagenesis to types of mutations and UV damage. Not cover these topics on pages 338-348 Errors in Replication Chemical modification by oxidation Chemical modification by alkylation Chemicals that cause deletions Ionizing radiation Cover all repair mechanisms Not cover these questions: Questions 7.1-7.6, 7.12, 7.15-7.16. [For questions 7.18 and 7.19a, refer to the answers to questions 7.15 and 7.16 to see the damaged DNA to be repaired.]

  2. Rest of Part Two Recombination: Chapter 8 Classes: October 11, 16 Transposition: Chapter 9 Class October 18 Exam: October 21

  3. October 07 class • Finish replication enzymes: 2_2_repl_enzy2.pdf • Primosome • Summarize origins, terminators: 2_3_ori_ter.pdf • Topological problems in replication: 2_4_telom_topo_reg.pdf • Telomerase • Topoisomerases • DNA supercoiling

  4. Primase • Synthesizes short oligonucleotides from which DNA polymerases can begin synthesis. • Combination of ribonucleotides and deoxyribonucleotides • Does not itself require a primer. • E. coli primase is DnaG, 60 kDa • Acts within a large primosome.

  5. Primers made by DnaG • Primers can be as short as 6 nt, as long as 60 nt. • Can substitute dNTPs for rNTPs in all except 1st and 2nd positions • Make hybrid primers with dNMPs and rNMPs interspersed. • Primase binds to CTG • T serves as template for 1st nucleotide of primer.

  6. Primosome has many proteins Pre-priming complex: Protein gene function PriA priA helicase, 3' to 5' movement, site recognition PriB priB PriC priC DnaT dnaT needed to add DnaB-DnaC complex to preprimosome DnaC dnaC forms complex with DnaB DnaB dnaB helicase, 5' to 3' movement, is a hexamer DNA dependent ATPase. Primase = DnaG

  7. Assay for assembly and migration of the primosome Convert single stranded (ss) fX174 to duplex, replicative form (RF)

  8. Steps in priming and synthesis

  9. Activities of DnaB and PriA in replisome “Sewing machine” model

  10. Control of DNA replication Replicon Origins and terminators Solutions to the “end problem” (telomeres) Cellular control mechanisms

  11. Oct 07 Class • Finish replication enzymes: 2_2_repl_enzy2.pdf • Primosome • Summarize origins, terminators: 2_3_ori_ter.pdf • Topological problems in replication: 2_4_telom_topo_reg.pdf • Telomerase • Topoisomerases • DNA supercoiling

  12. Replicon = unit that controls replication Replicator: cis-acting DNA sequence required for initiation; defined genetically Origin: site at which DNA replication initiates; defined biochemically Initiator: protein needed for initiation, acts in trans

  13. Theta-form replication intermediates visualized in EM for polyoma virus B. Hirt

  14. Labeling of completed DNA molecules can map replication origins Dana and Natahans, 1972, PNAS: map the replication origin of SV40 by labeling replicating molecules for increasing periods of time, isolating complete molecules, digesting with Hind restriction endonucleases, and determining which fragments have the most radioactivity.

  15. 2-D gels: map number & position of replication origins

  16. Positions of oriC and ter in E. coli Replication fork 2

  17. 245 bp long 4 copies of a 9 bp repeat 3 copies of a 13 bp repeat 11 GATC motifs Structure of oriC 13 13 13 9 9 9 9 1 GGATCCGGAT AAAACATGGT GATTGCCTCG CATAACGCGG TATGAAAATG GATTGAAGCC 61 CGGGCCGTGG ATTCTACTCA ACTTTGTCGG CTTGAGAAAG ACCTGGGATC CTGGGTATTA 121 AAAAGAAGAT CTATTTATTT AGAGATCTGTTCTATTGTGA TCTCTTATTA GGATCGCACT 181 GCCCTGTGGA TAACAAGGAT CCGGCTTTTA AGATCAACAA CCTGGAAAGG ATCATTAACT 241 GTGAATGATC GGTGATCCTG GACCGTATAA GCTGGGATCA GAATGAGGGG TTATACACAA 301 CTCAAAAACT GAACAACAGT TGTTCTTTGG ATAACTACCG GTTGATCCAA GCTTCCTGAC 361 AGAGTTATCC ACAGTAGATC GCACGATCTG TATACTTATT TGAGTAAATT AACCCACGAT

  18. Initiation at oriC: Model

  19. Termination and resolution

  20. Regulation of replication by methylation m m G A T C G A T C C T A G C T A G m m G A T C methylate replicate C T A G (lags m m G A T C G A T C behind replication) C T A G C T A G m m dam methylase Hemimethylated Fully methylated Fully methylated Will not replicate Will replicate Will replicate

  21. Oct 07 Class • Finish replication enzymes: 2_2_repl_enzy2.pdf • Primosome • Summarize origins, terminators: 2_3_ori_ter.pdf • Topological problems in replication: 2_4_telom_topo_reg.pdf • Telomerase • Topoisomerases • DNA supercoiling

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