Question

Question 3

(Adapted from Okazaki, R., et al. 1968. Proc Natl Acad Sci USA 59:598–605)

After the discovery of DNA polymerase by Arthur Kornberg, the properties of the enzyme became quite well known. One of the most critical is that all known DNA polymerases synthesize DNA only: 5' to 3'. This fact led to a dilemma regarding how the semiconservative model would work for a DNA molecule. Reiji Okazaki a brilliant experimenter took on this problem. [An aside: Okazaki was born near Hiroshima, Japan, in 1930. He was a teenager there at the time of the explosion of the first of two nuclear bombs that the US dropped at the end of World War II. Reiji's scientific career was cut short by his untimely death from cancer in 1975 at the age of 44]

In the actual experiment: They cultured E. coli (at low temperatures 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.

3a- 2pts) Describe what types of DNA fragments he would have observed after short pulses? Indicate in your answer:

1. What the difference is between the fragments in terms of size
2. Based on your understanding of the process of DNA replication what the source of those

fragments would be.

(Use accurate terminology. We will be using a Rubric that looks for the accurate use of terms needed to answer this question)

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.

3b- 1pt) What kinds of fragments would be seen after 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 seen after a long exposure to the radioactive label? (0.5pt) Why? (0.5pt) Answer in a complete sentence

Answer 1

3a the fragment that is observed should be radioactive and these fragments of DNA are also known as Okazaki fragments. Only Okazaki fragments are short pieces formed in DNA replication.

1. the difference between the fragment size is that since replication process is slowed down, there will be more large size fragment and less small size fragments as small size fragments cause replication

2. DNA polymerase is the source of those fragments.

3b. since there is a mutation in DNA ligase, this prevents the small segments of DNA. due to which the DNA remain short in size.

3c. the fragments seen are radiolabelled. this can cause mutation