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 (questions ) Cover all but “Cellular control of replication” pages , 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 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 , 7.12, [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.]

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

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

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.

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.

Primosome has many proteins 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. Pre-priming complex: Primase = DnaG

Assay for assembly and migration of the primosome Convert single stranded (ss)  X174 to duplex, replicative form (RF)

Steps in priming and synthesis

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

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

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

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

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

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.

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

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

Structure of oriC 245 bp long –4 copies of a 9 bp repeat –3 copies of a 13 bp repeat –11 GATC motifs GGATCCGGAT AAAACATGGT GATTGCCTCG CATAACGCGG TATGAAAATG GATTGAAGCC 61 CGGGCCGTGG ATTCTACTCA ACTTTGTCGG CTTGAGAAAG ACCTGGGATC CTGGGTATTA 121 AAAAGAAGAT CTATTTATTT AGAGATCTGT TCTATTGTGA 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

Initiation at oriC: Model

Termination and resolution

Regulation of replication by methylation G A T C C T A G m m G A T C C T A G m G A T C C T A G m G A T C C T A G m m G A T C C T A G m m replicate methylate (lags behind replication) Fully methylated Hemimethylated dammethylase Fully methylated Will replicate Will not replicateWill replicate

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