nullnull* 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 Replicationnull1) In synchronously replicating cells, density transfer experiments can reveal which sequences replicate earliest. What is the direction of fork movement here?null2) Electron microscopy of bubbles after restriction
enzyme digestionnullnull3) Hybridizing lagging strand nascent DNA to strand-specific fragments
shows site of switch from leading to lagging strand synthesisnull4) 2D Gel electrophoresis of replication intermediates followed by
hybridization with a DNA fragment reveals whether a replication
bubble originates in the fragmentDimension 1: separates by size; dimension 2: separates by shapenullSequence that when added to a non-replicating DNA causes it to
replicate; this approach was used to identify
Autonomously Replicating Sequences (ARS) in yeast. nullSequence that when mutated causes a replicating DNA to
fail to replicate; this approach was used to identify
essential elements in an (ARS) in yeastnullLinker scanning mutations in yeast ARS1% URA+ coloniesExamples of
mutantsAB1B2B3WT-ARS1nullnullOriC in E. coli chromosomal DNAnullInitiation of DNA replication in E. colinull 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 PrimaseDnaA and oriC independent replication initiation in E. coli
from hairpins, D-loops and R loopsnullSV40: a polyomavirus that is very useful for
studying eukaryotic replication enzymesnullEarly genes : proteins essential for viral DNA replicationnullT antigen binding sites cluster around the orinull* 98% nuclear: NLS
* origin-specific DNA binding
* DNA independent ATPase
* ATP dependent DNA helicase
* Binds to DNA polymerases, AP2, p53, Rb
* Undergoes phosphorylation etc.Properties of SV40 T antigennullLi & Kelly
SV40 DNA replication
In vitro.
PNAS (1984) 81:6973What else could they do
to validate their system?nullFractionation to homogeneitynullELONGATION FACTORS ARE CONSERVED
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