Question

The bacterial replication machinery has been isolated and examined in vitro. The best way to learn about the replication apparatus is to isolate it and them examine how each part works by itself and in concert with the other parts. Kornberg and coworkers worked very hard purifying all enzymes required for replication Initiation to try and study the process in detail. They then took the bacterial chromosome and digested it and plasmid DNA with restriction endonucleases (see below). They then ligated random regions of the bacterial chromosome into that of the plasmid to get a library of chromosomal fragments inserted into the plasmid below. Each of these plasmids were then subjected to in vitro replication, (a) In the blank space below, draw a picture that would represent what you would see in the three test tubes each containing one of the recombinant plasmids below and all of the proteins necessary for in vitro replication, (b) If you were to run each of those samples on an agarose gels what would you expect to see?

Answer 1

a)

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enzymes needed for invitro replication -

Dna B - helicase activity unwinds the DNA

Rep A - Binds with Dna B and and prometes the formation of replication fork

SSB - Single Standard Binding Proteins which binds single standard DNA and prevents the renaturation of DNA.

Primase - enzyme which synthesizes Primer

DNA Polymerase - involves in Polymerization process

DNA Gyrase - helpful in release of supercoil constraint

DNA ligase - seal Okazaki fragments

b) a thick band of Plasmid DNA observed on the gel

S+ 220 tube 3rd Tube