斯坦福大学分子生物学课件-Replication2A.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. Characteristics used to define Origins of 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 Dimension 1: separates by size; dimension 2: separates by shape 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 % URA+ colonies Examples of mutants A B1 B2 B3 WT-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 DnaA and oriC independent replication initiation in E. coli from hairpins, D-loops and R loops SV40: a polyomavirus that is very useful for studying eukaryotic replication enzymes *