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DNA replication

DNA replication. Semi-conservative mechanism. 1958, Meselson & Stahl 15 N labeling experiment. Rosalind Franklin (1920-1958) Maurice Wilkins (1916-2004). Francis Crick (1916-2004) James Watson (1928-). Discovery of DNA structure 1962 Nobel Prize.

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DNA replication

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  1. DNA replication Semi-conservative mechanism 1958, Meselson & Stahl 15N labeling experiment

  2. Rosalind Franklin (1920-1958) Maurice Wilkins (1916-2004) Francis Crick (1916-2004) James Watson (1928-) Discovery of DNA structure 1962 Nobel Prize

  3. The substrates of DNA synthesis dNTPs – dATP, dGTP, dCTP, dTTP Direction: 5’-3’

  4. T C A C OH ppp OH T C AC OH 3’ 5’ 3’ + 5’PPP 5’PPP + ppi

  5. G A T C G ppp OH + 5’ ppp OH 3’ GA T C G 5’ ppp OH 3’ 3’ 5’ ???

  6. A T C G G ppp OH + 5’ ppp OH 3’ G A T C G 5’ ppp ppp OH 3’ ???

  7. A T C G T C G A ppp OH ppp + p OH T C G T C G p OH pp p OH Proofreading??? pp

  8. 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

  9. All prokaryotic chromosomes and many bacteriophage and viral DNA molecules are circular and comprise single replicons. There is a single termination site roughly 180o opposite the unique origin.

  10. 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

  11. Semi-discontinuous replication Experimental evidences [3H] thymidine pulse-chase labeling experiment 1. Grow E. coli 2. Add [3H] thymidine in the medium for a few second,spin down and break the cell to stop labeling, analyze and find 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

  12. Back

  13. Bacterial DNA replication Experimental systems 1. Purified DNA: smaller and simpler bacteriophage and plasmid DNA molecules (ΦX174, 5 Kb) 2. All the proteins and other factors for its complete replications

  14. Initiation: oriC Study system the E. coli origin locus oriC is cloned into plasmids to produce more easily studied minichromosomes which behave like E.coli chromosome.

  15. 1. oriCcontains 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 attaches to the DNA and synthesizes a short RNA primer for synthesis of the leading strand. 6. Primosome DnaB helicase and DNA primase

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

  17. Elongation 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 α-subunit---polymerase ε-subunit---3’ 5’ proofreading exonuclease β-subunit---clamp the polymerase to DNA other subunits are different.

  18. 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. 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. Prokaryotic DNA replication

  19. Termination and segregation Terminus containing several terminator sites (ter) approximately 180o opposite oriC. Tus protein ter binding protein, an inhibitor of the DnaB helicase Topoisomerase IV a type II DNA topoisomerase, function to unlink the interlinked daughter genomes.

  20. Eukaryotic DNA replication Experimental systems 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): 14 Mb 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.

  21. Cell cycle

  22. Entry into the S-phase Cyclins CDKs (Cyclin-dependent protein kinases)

  23. DNA Replication DNA replication is semi-conservative, one strand serves as the template for the second strand. Furthermore, DNA replication only occurs at a specific step in the cell cycle. The following table describes the cell cycle for a hypothetical cell with a 24 hr cycle. Stage Activity Duration G1 Growth and increase in cell size 10 hr S DNA synthesis 8 hr G2 Post-DNA synthesis 5 hr M Mitosis 1 hr DNA replication has two requirements that must be met: 1. DNA template 2. Free 3' -OH group

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

  25. Electron Microscopy of replicating DNA reveals replicating bubbles.

  26. 3. Individual yeast replication origins (ARS) have been cloned into prokaryotic plasmids which allow these plasmids to replicate in yeast (an eukaryote). ARSs autonomously replicating sequences Minimal sequence 11 bp [A/T]TTTAT[A/G]TTT[A/T] (TATA box) 4. ORC (origin recognition complex) binds to ARS, upon activation by CDKs, ORC will open the DNA for replication.

  27. Elongation 1. Replication fork - unwinding DNA from nucleosomes: 50 bp/sec - need helicases and replication protein A (RP-A) - new nucleosomes are assembled to DNA from a mixture of old and newly synthesized histones after the fork passes

  28. 2. Elongation Three different DNA polymerases are involved 1) DNA pol α 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 δ on the leading strand that replaces DNA pol α., can synthesize long DNA 3) DNA pol ε on the lagging strand that replaces DNA pol α., synthesized Okazaki fragments are very short (135 bp in SV40), reflecting the amount of DNA unwound from each nucleosome.

  29. Nuclear matrix 1. A scaffold of insoluble protein fibers which acts as an organizational framework for nuclear processing, including DNA replication, transcription 2. Replication factories containing all the replication enzymes and DNA associated with the replication forks in replication BudR labeling of DNA

  30. Telomere replication

  31. Telomerase 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.

  32. cell dies or does not divide Telomerase activity is repressed in somatic cells of multicelluar organisms resulting in a gradual shortening of the chromosome with each cell generation. As this shortening reaches informational DNA, the cells senesce and die. When telomerase activityis repressed informational DNA cell division

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