Question

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

All known DNA polymerases can extend an existing strand in the 5'-3' direction. They can not initiate strand synthesis from the beginning. They can not extend the strand synthesis in 3'-5' direction.

3a. DNA replication is semiconservative and discontinuous. The leading strand is synthesized cond=tinuously in the 5'-3' direction while the lagging strand is also synthesized in 5'-3' direction discontinuously in the form of short Okazaki fragments. So, if the DNA replication is stopped after short pulses, Okazaki fragments can be isolated (As the replication progresses, Okazaki fragments are ligated into a single strand by the action of DNA ligase).

1. Okazaki fragments size in prokaryotes is 100-200 ntds.
Size of Okazaki fragments in eukaryotes is 1000-2000 ntds.

2. The small fragments on the gel correspond to Okazaki fragments synthesized on the lagging strand.

3b.
1. In the absence of DNA ligase, Okazaki fragments can not be ligated together to form a single continuous strand. So, we would observe short fragments on the gel after a short pulse.
2. In the absence of DNA ligase, Okazaki fragments can not be ligated together to form a single continuous strand even after a long pulse. So, we would still observe short Okazaki fragments.