After the discovery of DNA polymerase by Arthur Kornberg, theproperties of the enzyme became quite well known. One of the mostcritical is that all known DNA polymerases synthesize DNA only: 5'to 3'. This fact led to a dilemma regarding how thesemiconservative model would work for a DNA molecule. Reiji Okazakia brilliant experimenter took on this problem. [An aside: Okazakiwas born near Hiroshima, Japan, in 1930. He was a teenager there atthe time of the explosion of the first of two nuclear bombs thatthe US dropped at the end of World War II. Reiji's scientificcareer was cut short by his untimely death from cancer in 1975 atthe age of 44]
In the actual experiment: They cultured E. coli (at lowtemperatures to slow down replication process) in the presence ofradioactive nucleotides. They did this for short pulses followed bythe addition of excess nonradioactive nucleotides. This resulted inlabel (radioactivity) being present only in the DNA that wassynthesized during the short period of the pulse. Soon after thepulse, they isolated the DNA and separated the individual strandsfrom one another. The various pieces of DNA could then be sortedout by size: using a “sucrose gradient†and spun in anultracentrifuge.
3a- 2pts) Describe what types of DNA fragmentshe would have observed after short pulses? Indicate in youranswer:
1. What the difference is between the fragments in terms ofsize
2. Based on your understanding of the process of DNA replicationwhat the source of those
fragments would be.
(Use accurate terminology. We will be using a Rubric thatlooks for the accurate use of terms needed to answer thisquestion)
Assume Okazaki and his team were unaware that their bacteria hada mutation in the gene that codes for DNA Ligase. Also assume thatthe mutation rendered the protein DNA Ligase unable to carry outits enzymatic activity/function.
3b- 1pt) What kinds of fragments would be seenafter a short pulse when carrying out the assay with such a mutant?(0.5pt) Why? (0.5pt) (Answer in complete sentences)
3c- 1pt) What kind of fragments would be seenafter a long exposure to the radioactive label? (0.5pt) Why?(0.5pt) Answer in a complete sentence
