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Section E DNA Replication Molecular Biology Course.

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Presentation on theme: "Section E DNA Replication Molecular Biology Course."— Presentation transcript:

1 Section E DNA Replication Molecular Biology Course

2 E3: Eukaryotic DNA replication Molecular Biology Course E1: DNA Replication: An Overview Replicons, semi-conservative, semi- discontinous, RNA priming E2: Bacterial DNA replication Experimental system, initiation, unwinding, elongation, termination & segregation Experimental system, cell cycle, initiation, replication forks, nuclear matrix, telomere repl.

3 DNA replication E1: DNA Replication: An Overview 1.Replicons 2.semi-conservative mechanism 3.semi-discontinous replication 4.RNA priming

4 Replicon is any piece of DNA which replicates as a single unit. It contains an origin and sometimes a terminus Origin is the DNA sequence where a replicon initiates its replication. Terminus is the DNA sequence where a replicon usually stops its replication DNA replication E1-1 Replicons

5 Prokaryotic genome: a single circular DNA = a single replicon Eukaryotic genome: multiple linear chromosomes & multiple replicons on each chromosome DNA replication

6 Bidirectional replication of a circular bacterial replicon All prokaryotic chromosomes and many bacteriophage and viral DNA molecules are circlular and comprise single replicons. There is a single termination site roughly 180 o opposite the unique origin. DNA replication

7 Linear viral DNA molecules usually have a single origin, replication details (see Section R) In all the cases, the origin is a complex region where the initiation of DNA replication and the control of the growth cycle of the organism are regulated and co-ordinated. DNA replication

8 The long, linear DNA molecules of eukaryotic chromosomes consist of mutiple regions, each with its own orgin. A typical mammalian cell has 50000-100000 replicons with a size range of 40-200 kb. When replication forks from adjacent replication bubbles meet, they fuse to form the completely replicated DNA. No distinct termini are required Multiple eukaryotic replicons and replication bubbles DNA replication

9 replication bubbles  replication fork See Page 74 of your text book

10 DNA replication E1-2 Replication is Semi-conservative

11 Semi-conservative mechanism 15 N labeling experiment 1. 15 N labeling: grow cells in ?? 2.Collect DNA: grow cells in ?? 3.Separation: method ?? 4.Result interpretation 15 N labeled DNA unlabeled DNA DNA replication


13 E1-3 Replication is Semi-discontinuous

14 Semi-discontinuous replication Ligation DNA replication Okazaki fragments

15 Discovery of Okazaki fragments Evidence for semi-discontinuous replication [ 3 H] thymidine pulse-chase labeling experiment 1.Grow E. coli 2.Add [ 3 H] thymidine in the medium for a few second  spin down and break the cell to stop labeling  analyze  found a large fraction of nascent DNA (1000-2000 nt) = Okazaki fragments 3.Grow the cell in regular medium then  analyze  the small fragments join into high molecular weight DNA = Ligation of the Okazaki fragments DNA replication

16 E1-4 RNA priming The first few nucleotides at the 5’-end of Okazaki fragments are ribonucleotides. Hence, DNA synthesis is primed by RNA that is then removed before fragments are joined. Crucial for high fidelity of replication

17 DNA replication E2: Bacterial DNA replication E2: Bacterial DNA replication 1.Experimental system 2.initiation, 3.unwinding, 4.elongation, 5.termination & segregation

18 E2-1: In vitro experimental systems 1.Purified DNA: smaller and simpler bacteriophage and plasmid DNA molecules (  X174, 5 Kb) 2.All the proteins and other factors for its complete replications DNA replication In vitro system: Put DNA and protein together to ask for replication question

19 Study system : the E. coli origin locus oriC is cloned into plasmids to produce more easily studied minichromosomes which behave like E. coli chromosome. DNA replication E2-2: Initiation

20 1.oriC contains four 9 bp binding sites for the initiator protein DnaA. Synthesis of DnaA is coupled to growth rate so that initiation of replication is also coupled to growth rate. 2.DnaA forms a complex of 30-40 molecules, facilitating melting of three 13 bp AT-rich repeat sequence for DnaB binding. 3.DnaB is a helicase that use the energy of DNA hydrolysis to further melt the double-stranded DNA. 4.Ssb (single-stranded binding protein) coats the unwinded DNA. 5.DNA primase load to synthesizes a short RNA primer for synthesis of the leading strand. 6.Primosome: DnaB helicase and DNA primase

21 Initiation

22 Re-initiation of bacterial replication at new origins before completion of the first round of replication

23 Positive supercoiling: caused by removal of helical turns at the replication fork. Resolved by a type II topoisomerase called DNA gyrase DNA replication E2-3: Unwinding

24 DNA replication E2-4: Elongation

25 DNA polymerase III holoenzyme: 1.a dimer complex, one half synthesizing the leading strand and the other lagging strand. 2.Having two polymerases in a single complex ensures that both strands are synthesized at the same rate 3.Both polymerases contain an  -subunit---polymerase  -subunit---3’  5’ proofreading exonuclease  -subunit---clamp the polymerase to DNA other subunits are different. Replisome : in vivo, DNA polymerase holoenzyme dimer, primosome (helicase) are physically associated in a large complex to synthesize DNA at a rate of 900 bp/sec.


27 Other two enzymes during Elongation 1. Removal of RNA primer, and gap filling with DNA pol I 2. Ligation of Okazaki fragments are linked by DNA ligase.

28 Elongation: lagging strand replication Polymerase III holoenzyme (DNA pol III) DNA pol I (5’  3’ exonulclease activity) DNA pol I (5’  3’ polymerase activity) DNA ligase

29 DNA replication E2-5: Termination and Segregation

30 Terminus: containing several terminator sites (ter) approximately 180 o opposite oirC. Tus protein: ter binding protein, an inhibitor of the DnaB helicase Termination ter

31 Topoisomerase IV: a type II DNA topoisomerase, function to unlink the interlinked daughter genomes. Segregation

32 DNA replication E3: Eukaryotic DNA replication E3: Eukaryotic DNA replication 1.Experimental system 2.cell cycle, 3.initiation, 4.replication forks, 5.nuclear matrix, 6.telomere replication.

33 E3-1: In vitro experimental systems 1.Purified DNA : 2.All the proteins and other factors for its complete replications DNA replication

34 1.Small animal viruses (simian virus 40, 5 kb) are good mammalian models for elongation (replication fork) but not for initiation. 2.Yeast (Saccharomyces cerevisiae): 1.4 X 10 7 bp in 16 chromosomes, 400 replicons, much simpler than mammalian system and can serve as a model system 3.Cell-free extract prepared from Xenopus (frog) eggs containing high concentration of replication proteins and can support in vitro replication.

35 E3-2: Cell cycle When to replicate DNA replication

36 G1 preparing for DNA replication (cell growth) S DNA replication G2 a short gap before mitosis M mitosis and cell division Cell cycle Entry into the S-phase: Cyclins Cyclin-dependent protein kinases (CDKs) signaling

37 E3-2: Iniation of multiple replicons DNA replication 1.Timing 2.Order

38 1.Clusters of about 20-50 replicons initiate simultaneously at defined times throughout S- phase Early S-phase: euchromatin replication Late S-phase: heterochromatin replication Centromeric and telomeric DNA replicate last

39 2. Only initiate once per cell cycle Licensing factor: required for initiation and inactivated after use Can only enter into nucleus when the nuclear envelope dissolves at mitosis

40 Licensing factor Initiation

41 Initiation: origin 1.Yeast replication origins (ARS- autonomously replicating sequences, enables the prokaryotic plasmids to replicate in yeast). Minimal sequence of ARS: 11 bp [A/T]TTTAT[A/G]TTT[A/T] (TATA box) Additional copies of the above sequence is required for optimal efficiency. 2.ORC (origin recognition complex) binds to ARS, upon activation by CDKs, ORC will open the DNA for replication.

42 E3-3: Replication fork & elongation DNA replication 1.unwinding 2.enzymes

43 Replication fork Unwinding DNA from parental nucleosomes before replication : 50 bp/sec, helicases and RP-A New nucleosomes are assembled to DNA from a mixture of old and newly synthesized histones after the fork passes.

44 Elongation: three different DNA polymerases are involved. 1.DNA pol  : contains primase activity and synthesizes RNA primers for the leading strands and each lagging strand fragments. Continues elongation with DNA but is replaced by the other two polymerases quickly. 2.DNA pol  : on the leading strand that replaces DNA pol . can synthesize long DNA 3.DNA pol  : on the lagging strand that replaces DNA pol  synthesized Okazaki fragments are very short (135 bp in SV40), reflecting the amount of DNA unwound from each nucleosome.

45 E3-4: Nuclear Matrix DNA replication A scaffold of insoluble protein fibers which acts as an organizational framework for nuclear processing, including DNA replication, transcription

46 Replication factories: all the replication enzymes, DNA associated with the replication forks in replication BUdR labeling of DNA Visualizing by immunoflurescence using BUdR antiboby

47 E3-3: Telomere replication DNA replication Solving the problem of lagging strand synthesis -- Chromosomal ends shortening 5’ 3’ 5’3’ 5’ 3’ 5’ 3’ 5’ 3’ Parental DNA Daughter DNAs

48 telomerase DNA replication

49 1.Contains a short RNA molecule as telomeric DNA synthesis template 2.Telomerase activity is repressed in the somatic cells of multicellular organism, resulting in a gradual shortening of the chromosomes with each cell generation, and ultimately cell death (related to cell aging) 3.The unlimited proliferative capacity of many cancer cells is associated with high telomerase activity. Telomerase DNA replication See movie for cancer metastasis

50 DNA polymerase control the fidelity of DNA replication Proofreading refers to any mechanism for correcting errors in protein or nucleic acid synthesis that involves scrutiny of individual units after they have been added to the chain Processive DNA polymerases have 3’  5’ exonuclease activity Supplemental 1

51 by E. coli polymerase Proofreading Supplemental 2

52 Crystal structure of phage T7 DNA polymerase Exonuclease domain template

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