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* The position on the DNA at which replication start points are found. (4 methods discussed)

Characteristics used to define Origins of Replication. * The position on the DNA at which replication start points are found. (4 methods discussed) * A DNA sequence that when added to a non-replicating DNA causes it to replicate. * A DNA sequence whose mutation abolishes replication.

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* The position on the DNA at which replication start points are found. (4 methods discussed)

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  1. Characteristics used to define Origins of Replication * The position on the DNA at which replication start points are found. (4 methods discussed) * A DNA sequence that when added to a non-replicating DNA causes it to replicate. * A DNA sequence whose mutation abolishes replication. * A DNA sequence that in vitro is the binding target for enzyme complexes known to function in initiation of DNA replication. 免疫学信息网http://www.immuneweb.com

  2. 1) In synchronously replicating cells, density transfer experiments can reveal which sequences replicate earliest. What is the direction of fork movement here? 免疫学信息网http://www.immuneweb.com

  3. 2) Electron microscopy of bubbles after restriction enzyme digestion 免疫学信息网http://www.immuneweb.com

  4. 免疫学信息网http://www.immuneweb.com

  5. 3) Hybridizing lagging strand nascent DNA to strand-specific fragments shows site of switch from leading to lagging strand synthesis 免疫学信息网http://www.immuneweb.com

  6. 4) 2D Gel electrophoresis of replication intermediates followed by hybridization with a DNA fragment reveals whether a replication bubble originates in the fragment Dimension 1: separates by size; dimension 2: separates by shape 免疫学信息网http://www.immuneweb.com

  7. Sequence that when added to a non-replicating DNA causes it to replicate; this approach was used to identify Autonomously Replicating Sequences (ARS) in yeast. 免疫学信息网http://www.immuneweb.com

  8. Sequence that when mutated causes a replicating DNA to fail to replicate; this approach was used to identify essential elements in an (ARS) in yeast 免疫学信息网http://www.immuneweb.com

  9. Linker scanning mutations in yeast ARS1 Examples of mutants WT-ARS1 B3 B2 B1 A % URA+ colonies 免疫学信息网http://www.immuneweb.com

  10. 免疫学信息网http://www.immuneweb.com

  11. OriC in E. coli chromosomal DNA 免疫学信息网http://www.immuneweb.com

  12. Initiation of DNA replication in E. coli 免疫学信息网http://www.immuneweb.com

  13. DnaA and oriC independent replication initiation in E. coli from hairpins, D-loops and R loops Two primosomes for E. coli chromosomal replication Site of primosome assembly oriC, A-site n'-pas, D-loop, R-loop Recognition DnaA protein PriA protein Auxilliary proteins for (HU for oriC) PriB loading helicase PriC DnaT Helicase DnaB/DnaC Priming Primase 免疫学信息网http://www.immuneweb.com

  14. SV40: a polyomavirus that is very useful for studying eukaryotic replication enzymes 免疫学信息网http://www.immuneweb.com

  15. Early genes : proteins essential for viral DNA replication 免疫学信息网http://www.immuneweb.com

  16. T antigen binding sites cluster around the ori 免疫学信息网http://www.immuneweb.com

  17. Properties of SV40 T antigen * 98% nuclear: NLS * origin-specific DNA binding * DNA independent ATPase * ATP dependent DNA helicase * Binds to DNA polymerases, AP2, p53, Rb * Undergoes phosphorylation etc. 免疫学信息网http://www.immuneweb.com

  18. Li & Kelly SV40 DNA replication In vitro. PNAS (1984) 81:6973 What else could they do to validate their system? 免疫学信息网http://www.immuneweb.com

  19. Fractionation to homogeneity 免疫学信息网http://www.immuneweb.com

  20. ELONGATION FACTORS ARE CONSERVED 免疫学信息网http://www.immuneweb.com

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