Biol 3054
Worksheet 5
1. You have recently discovered a new protozoan (a single celled eukaryote) from deep sea vent. You are very interest in studying DNA replication in this
organism since it lives at temperatures in excess of 100C. You grew some of the organisms in the lab in the presence of varying concentrations of radioactively
labeled deoxynucleotides to visualize DNA replication. The experiment was as follows:
1. grew for 5 minutes in low concentrations of 32P-dTTP
2. added remaining radiolabeled dNTPs at increased concentrations and allowed cells to grow another 5 minutes.
3. formaldehyde rixed the cells to halt DNA replication
4. exposed the cells to a special photographic emulsion and obtained this photomicrograph of replicating DNA.
Interpret these results as was done with the similar experiments as shown and discussed in lecture. In particular, how does DNA replication in this new organism
compare to DNA replication in other known eukaryotic organsims? Neatly write your answer on this page.
The experimental results shown and discussed in lecture showed a pattern where the least radioactivity was located centrally and more incorporation was located
laterally in a head to head manner as shown here:
which indicated that DNA synthesis was occuring bidirectionally from a central
origin of replication. The head to tail pattern seen with the experimental results from the new protozoan indicate that DNA replication is occuring in a
unidirectional manner. This is very different from the mechanism to be operating in known eukaryotes.
Biol 3054
Worksheet 5
2. Replication of the E. coli genome (4.5x106bp) takes 15 minutes. How many nucleotides/min does E. coli DNA pol III polymerize? Show your math work
and circle your final answer.
Since DNA synthesis is bidirectional, ½ of the total genome is replicated in 15 min. Therefore, the rate of DNA synthesis by pol III is 2.25x106 nt/15 min or
1.5x105 nt/min or 2500nt/sec.
3. Briefly define the roles of each of these proteins/enzymes in the replication process (use the space provided)
a. DNA Ligase – the enzyme which joins the ends of DNA strands together. Its activity is needed to join successive Okazaki fragments of the
lagging strand.
b. -subunit of E. coli DNA pol III – this subunit has the main polymerase activity of the holoenzyme – it actually incorporates nucleotides into
the growing DNA strand.
c. - subunit of E. coli DNA pol III – this subunit has the proofreading activity of the holoenzyme.
d. E. coli DNA pol I – this enzyme removes the RNA primers and replaces the nucleotides with deoxynucleotides.
e. -clamp – this protein secures the DNA pol III polymerase subunits onto the DNA template.
f. -complex of E. coli DNA pol III – these enzymes open the -clamp protein and lock it onto the DNA template and help it connect to the
DNA pol III polymerase subunits.
g. helicase – this enzyme unwinds the parental DNA double helix ahead of the replication forks
h. DNA gyrase – this enzyme cuts the DNA strands to allow them to unwind to relieve over winding stresses resulting form the twisting of the
replication process
i. primase – this enzyme synthesizes a short RNA primer complementary to the template DNA to provide a 3’OH for the synthesis of DNA by
DNA pol III.
Biol 3054
Worksheet 5
4. In the space below, diagram a replisome. Include all the enzymes, both parental and nascent DNA strands indicating their 5’3’ orientations,
and any other crucial features (Okazaki frags, primers etc..). The parts have to be labeled neatly and legibly.
Biol 3054
Worksheet 5
5. Draw a cell undergoing meiosis in a diploid organism that has a total of 8 chromosomes (n=4; 2n=8). Starting with a single cell, draw all the resulting cells and
all necessary components to demonstrate the meiotic process for ALL of the meiotic stages. Use this page for the entire diagram. Label as necessary.
Biol 3054
Worksheet 5
6. Describe how the cell cycle is controlled by the cyclins and cdk proteins.
Cyclin proteins bind to the Cdks (cyclin dependent kinases) and activate the Cdks’ kinase activity. The activated cdks, phosphorylate (kinase) proteins in the cell
that permit the cell to continue or enter the cell cycle. The cyclin proteins are specifically synthesized and then degraded during the various subdivisions of the
cell cycle. There are specific cyclin proteins produced during each phase of the cell cycle (M, G1, S, G2), each binds to specific sets of Cdks and stimulates the
their activity. During G1, for example, cylin D/Cdk4 and cyclin E/cdk2 phosphorylate the Rb protein to allow passage into S phase.
7. Describe the sequentially increasing levels of DNA packaging that occur in eukaryotic nuclei.
In an eukaryotic nucleus, the DNA of each chromosomes is a very long double stranded molecule, that if fully extended would be thousands of time longer than
the cell itself. To fit this DNA into the nucleus, it is packaged, which reduces its overall length. Double stranded DNA is first packaged by wrapping around a
core of 8 histone proteins to form nucleosomes. Successive nucleosomes are connected and form a coiled structure approximately 30nm in diameter . This 30nm,
coiled fiber is further looped and the loops gathered to a central core of proteins to form the arms of the chromosomes as viewed during mitosis (or meiosis).