Question

In the experiment, Okazaki cultured E. coli (at low temperatues to slow down replication process) in the presence of radioactive nucleotides. They did this for short pulses followed by the addition of excess nonradioactive nucleotides. This resulted in label (radioactivity) being present only in the DNA that was synthesized during the short period of the pulse. Soon after the pulse, they isolated the DNA and separated the individual strands from one another. The various pieces of DNA could then be sorted out by size: using a "sucrose gradient" and spun in an ultracentrifuge.

Assume Okazaki and his team were unaware that their bacteria had a mutation in the gene that codes for DNA ligase. Also assume that the mutation rendered the protein DNA Ligase unable to carry out its enzymatic activity/function.

1. What kinds of fragments would be seen after a short pulse when carrying out the assay with such a mutant? Why?

2. What kind of fragments would be seen after a long exposure to the radioactive label? Why?

Answer 1

1- As we know that DNA replication takes place in 5' to 3' direction. So when we give the short pulse there will be one long fragment and very little short fragment. the long fragment is the DNA fragment that is synthesized on the leading strand whereas short fragment will be synthesised on the lagging strand.

In case of long pulse, we will get one long fragment and many short fragments. the short fragments are the one which is synthesized on the lagging standard template. As we know that lagging strand is synthesized only when the short stretch of DNA is opened. So when the long pulse is given we will get so many shote fragments