Chapter 9 Replication of DNA Chapter 9 Opener

DNA synthesis requires Figure 9-1 DNA synthesis requires Figure 9-1 DNA synthesis requires 2

Figure 9-1a Figure 9-1a 3

Figure 9-1b DNA is synthesized by extending the 3’ end of the primer (made of RNA by primase) Figure 9-1b DNA is synthesized by extending the 3’ end of the primer 4

Figure 9-2 Figure 9-2 5

DNA polymerases use a single active site toatalyze Figure 9-3 DNA polymerases use a single active site toatalyze Figure 9-3 DNA polymerases use a single active site to catalyze 6

Steric constraints (입체적 장해) prevent DNA Pol from using rNTP Figure 9-4 Steric constraints (입체적 장해) prevent DNA Pol from using rNTP Figure 9-4 Steric constraints prevent DNA Pol from using rNTP 7

Figure 9-5 Figure 9-5 8

Two metal ions bind to DNA Polymerase catalyze nucleotide addition Figure 9-6 Two metal ions bind to DNA Polymerase catalyze nucleotide addition Figure 9-6 Two metal ions bind to DNA Polymerase catalyze nucleotide addition 9

Figure 9-6a Figure 9-6a 10

Figure 9-6b Figure 9-6b 11

Figure 9-7 DNA polymerase grips template and incoming nucleotide when a correct base pair is made. Figure 9-7 DNA polymerase grips template and incoming nucleotide when a correct base pair is made. 12

Figure 9-7a Figure 9-7a 13

Figure 9-7b Figure 9-7b 14

O-helix Base and dNTP Primer Figure 9-7c Figure 9-7c O-helix Primer 15

Figure 9-8 Figure 9-8 16

Figure 9-9 Figure 9-9 17

Incorrect base is removed by proofreading exonuclease. Figure 9-10 Incorrect base is removed by proofreading exonuclease. (c.f. endonuclease) Figure 9-10 Incorrect base is removed by proofreading exonulcease. 18

Figure 9-11a Figure 9-11a 19

Mismatched 3-4 nucleotide exposed to exonuclease Figure 9-11b Mismatched 3-4 nucleotide exposed to exonuclease (DNA Pol itself) Figure 9-11b Mismatched 3-4 nucleotide exposed to exonuclease (DNA Pol itself) 20

Figure 9-11c Figure 9-11c 21

Figure 9-12 Replication fork Figure 9-12 Replication fork 22

Primase is a specialized RNA polymerase dedicated to make short RNA primers(5-10 nuleotides long) Primase initiate RNA synthesis using an ssDNA template of trimer (GTA in case of E. coli) when helicase unwinds dsDNA at the replication fork. Primase is a specialized RNA polymerase dedicated to make short RNA primers(5-10 nuleotides long)

Figure 9-13 Removal of RNA primer Figure 9-13 24

Figure 9-14 DNA helicase separate two strands of double helix before replication fork has 5’-3’ polarity Figure 9-14 DNA helicase (have 5’-3’ polarity) separate two strands of double helix before replication fork 25

Central channel Hexameric helicase Phosphate of ss DNA Figure 9-15 Central channel Hexameric helicase Phosphate of ss DNA Figure 9-15 Hexameric helicase Central channel Two hairpin structure Phosphate Two hairpin structure 26

Figure 9-15a Figure 9-15a 27

Figure 9-15b Figure 9-15b 28

Figure 9-16 Binding of SSB to DNA Figure 9-16 Binding of SSB to DNA. Only after SSWbs bind to completely coated initially bound ssDNA does bind occur on other molecules. Only after SSBs bind to completely coated initially bound ssDNA, does binding occur on other molecules. 29

Action of topoisomerase at the replication fork Figure 9-17 Action of topoisomerase at the replication fork Figure 9-17 Action of topoisomerase at the replication fork 30

Table 9-1 Table 9-1 31

(Specialization of DNA polymerases) Table 9-2 (Specialization of DNA polymerases) Table 9-2 Specialization of DNA polymerases 32

DNA polymerase switching during eukaryotic DNA synthesis Figure 9-18 DNA polymerase switching during eukaryotic DNA synthesis DNA Pol α has low processivity and switched with DNA Pol delta. Figure 9-18 DNA polymerase switching during eukaryotic DNA synthesis 33

Figure 9-19 With assistance of sliding clamp, DNA polymerase delta increases processivity(fast replication). Figure 9-19 With assistance of sliding clamp, DNA polymerase delta increases processivity. 34

Slow replication Normal replication Figure 9-20 Figure 9-20 35

Eukaryotic sliding camp(PCNA) T4 phage Sliding camp Figure 9-21 E. coli sliding camp Figure 9-21 E. coli sliding camp T4 phage Eukaryotic (PCNA) Eukaryotic sliding camp(PCNA) T4 phage Sliding camp 36

ATP control protein function: Loading a sliding camp Box 9-3-1 DNA pol ATP control protein function: Loading a sliding camp Box 9-3-1 ATP control protein function: Loading a sliding camp DNA pol 37

Composition of DNA Pol III holoenzyme Figure 9-22 Composition of DNA Pol III holoenzyme Figure 9-22 Composition of DNA Pol III holoenzyme 38

Figure 9-23 “Trombone” model for coordinating replication by two DNA polymerase s at E. coli replication fork. Figure 9-23 “Trombone” model for coordinating replication by two DNA polymerase s at E. coli replication fork. 39

Figure 9-23a Figure 9-23a 40

Figure 9-23b Figure 9-23b 41

Figure 9-23c Figure 9-23c 42

Figure 9-23d Figure 9-23d 43

Figure 9-23e Figure 9-23e 44

Figure 9-24a Binding of DNA helicase to DNA pol III holoenzyme stimulate the rate of DNA strand separation Figure 9-24a Combination of all of the proteins than function at the replication fork referred to as REPLISOME(DNA pol, Helicase, Primase) Combination of all of the proteins than function at the replication fork referred to as REPLISOME(DNA pol, Helicase, Primase) 45

Figure 9-24b Figure 9-24b 46

(particular origin of replication) model Figure 9-25 Initiation of DNA replication: specific genomic DNA sequence direct the initiation Replicon (particular origin of replication) model Figure 9-25 Initiation of DNA replication: specific genomic DNA sequence direct the initiation Replicom model 47

Structure of replicator Figure 9-26 Structure of replicator DNA element that facilitate DNA unwinding (blue) Initiator binding site (green) Figure 9-26 Structure of replicator DNA element that facilitate DNA unwinding (green) Site of initial DNA synthesis Site of initial DNA synthesis (red) 48

Figure 9-27 Figure 9-27 49

Figure 9-27a Figure 9-27a 50

Figure 9-27b Figure 9-27b 51

Figure 9-27c Figure 9-27c 52

Figure 9-27d Figure 9-27d 53

Figure 9-27e Figure 9-27e 54

Figure 9-27f Figure 9-27f 55

Figure 9-27g Figure 9-27g 56

Figure 9-27h Figure 9-27h 57

Eukaryotic chromosomes are replicated exactly once per cell cycle. Figure 9-28 Eukaryotic chromosomes are replicated exactly once per cell cycle. Figure 9-28 Eukaryotic chromosomes are replicated exactly once per cell cycle. Chromosome breakage as a results of incomplete DNA replication Chromosome breakage as a results of incomplete DNA replication 58

Replicators (of daughter DNA) are inactivated by DNA replication Figure 9-29 Replicators (of daughter DNA) are inactivated by DNA replication Replicator 1 is not reached by adjacent fork before initiation and normally initiated. Figure 9-29 Replicator 1 is not reached by adjacent fork before initiation and normally initiated. No replication in daughter 59

Eukaryotic helicase loading Figure 9-30 ORC: origin recognition complex Eukaryotic helicase loading Figure 9-30 ORC: origin recognition complex 60

Figure 9-31 Binding of Sld2, Sld3, Dpb11 facilitate binding of Cdc45 and Gins Kinase Helicase activating protein Activation of loaded helicases leads to assembly of eukaryotic replisome Figure 9-31 Activation of loaded helicases leads to assembly of eukaryotic replisome Kinase Binding of Sld2, Sld3, Dpb11 facilitate binding of Cdc45 and Gins Helicase activating protein (Cdc45 and Gins) 61

Helicase activation alters helicase interaction Figure 9-32 Helicase activation alters helicase interaction Figure 9-32 Cdc45-Mcm2-7-Gins (CMG) Helicase activation alters helicase interaction Cdc45-Mcm2-7-Gins (CMG) 62

Figure 9-33 In each cell cycle, only one opportunity for helicase to load on origins. Figure 9-33 In each cell cycle, only one opportunity for helicase to load on roigins. 63

ORC: origin recognition complex Figure 9-34 ORC: origin recognition complex Figure 9-34 64

Topoisomerase II catalyzes decatenation of replication products Figure 9-35 Topoisomerase II catalyzes decatenation of replication products Figure 9-35 Topoisomerase II catalyzes decatenation of replication products 65

Topoisomerase II catalyzes decatenation of replication products Figure 9-36 Topoisomerase II catalyzes decatenation of replication products Figure 9-36 Topoisomerase II catalyzes decatenation of replication products 66

Figure 9-37 Protein priming as a solution to end replication problem (in linear chromosome of certain bacterial species and viruses) Figure 9-37 Protein priming as a solution to end replication problem (in linear chromosome of certain bacterial species and viruses) 67

Replication of telomeres by telomerase in eukaryotes Figure 9-38 Replication of telomeres by telomerase in eukaryotes Head to tail of TG rich DNA sequences Figure 9-38 Replication of telomeres by telomerase Head to tail of TG rich DNA sequences 68

Figure 9-39 Figure 9-39 69

Telomere binding proteins Figure 9-40 Telomere binding proteins Yeast Human Figure 9-40 Telomere binding proteins Yeast Human 70

Telomere length regulation by telomere binding proteins Figure 9-41 Telomere length regulation by telomere binding proteins Figure 9-41 Telomere length regulation by telomere binding proteins 71

Telomere binding proteins protect chromosome ends Figure 9-42 Telomere binding proteins protect chromosome ends Figure 9-42 Telomere binding proteins protect chromosome ends 72

Figure 9-42b Figure 9-42b 73