DNA复制3幻灯片.pptVIP

* 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. ;1) In synchronously replicating cells, density transfer experiments can reveal which sequences replicate earliest. What is the direction of fork movement here?;2) Electron microscopy of bubbles after restriction enzyme digestion;;3) Hybridizing lagging strand nascent DNA to strand-specific fragments shows site of switch from leading to lagging strand synthesis;4) 2D Gel electrophoresis of replication intermediates followed by hybridization with a DNA fragment reveals whether a replication bubble originates in the fragment;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. ;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;Linker scanning mutations in yeast ARS1;;OriC in E. coli chromosomal DNA;Initiation of DNA replication in E. coli; 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;SV40: a polyomavirus that is very useful for studying eukaryotic replication enzymes;Early genes : proteins essential for viral DNA replication;T antigen binding sites cluster around the ori;* 98% nuclear: NLS * origin-specific DNA binding * DNA independent ATPase * ATP dependent DNA helicase * Binds to DNA polymerases, AP2, p53, Rb * Undergoes phosphorylation etc.;Li Kelly