1. The following terms refer to activities, enzymes, proteins, etc. that are involved in the DNA replication process. Next to the term, describe what role is played in DNA replication by that term. (3 pts each)

a. 3’-5’ exonuclease activity

Proofreading function. Removes an incorrect nucleotide immediately after it is inserted in the growing daughter strand that during DNA replication

b. topoisomerase

Relieves torsional strain (supercoiling) upstream from the replication fork caused by melting of the hydrogen bonds as the fork progresses. Does this by nicking one strand, allowing the other strand to swivel through the nick, and then repairing the nick, thus relieving the strain.

c. helicase

Melts the hydrogen bonds holding the ds DNA molecule together to allow for the parental strands to be used as a template during replication.

d. ligase

Creates the last phosphodiester linkage that connects Okazaki fragments to one another. This connection is not templated and therefore cannot be made by the DNA polymerase.

e. primase

Makes the short RNA primer that must be used to start all new DNA polymers during DNA replication. Operates during initiation of DNA replication and when every Okazaki fragment is begun.

f. 5’-3’ exonuclease activity

Removes the RNA primers so that DNA nucleotides can replace them.
 

2. Describe all steps in the excision repair pathway used by cells to repair thymine dimers. (8 pts)

See page 479, Figure 12-26

1. Two UvrA and one UvrB complex and move along the DNA until they encounter a lesion (dimmer).
2. ATP is hydrolyzed and the DNA is kinked in the region containing the lesion by 130 degrees.
3. UvrA dissociates and UvrC endonuclease binds and cuts the damaged strand at two sites on either side of the lesion.
4. UvrB and UvrC leave and helicase unwinds the damaged region, releasing the damaged region as a single strand of DNA which will be degraded.
5. DNA polymerase replaces the missing nucleotides and ligase reseals the last phosphodiester bond.
 

3. Dan is walking down a dark street one night and sees a suspicious person approaching. His heart starts beating quickly and his impulse is to run. In Dan’s cells at this time, glycogen synthase, an enzyme involved in synthesizing glycogen using glucose monomers, becomes inhibited. This is due to phosphorylation by the cyclic-AMP dependent protein kinase. Describe the signal transduction pathway that is working during this time in Dan that causes this phosphorylation to occur. (10 pts)

See page 867, Fig. 20-16; pages 884 and 885; and page 75, Fig. 3-27

A hormone is released in response to the fear. It binds to a receptor in Dan’s cells. The receptor changes shape and binds to a G protein called G stimulatory. This induces the alpha subunit to release GDP and GTP replaces it. The beta and gamma subunits are then unable to remain bound to the alpha subunit, and they release from it. The alpha subunit (still bound to GTP) binds to adenylyl cyclase in the membrane and activates it to catalyze the formation of cyclic AMP from ATP. The increased concentration of cyclic AMP causes it to interact with the cyclic AMP dependent protein kinase. When cyclic Amp binds to the regulatory subunits of this enzyme, They release the catalytic subunits. The catalytic subunits can now phosphorylate target molecules. Glycogen synthase is one of these target molecules.
 

4. This is a 2 part question about receptor tyrosine kinase signaling.

A. Illustrate all interactions from the activated receptor to the activation of the ras protein. In your illustration, circle the location of the SH2 domain, the SH3 domain, and the GEF. (10 pts)

See page 874, Fig. 20-23
 

B. Illustrate how the active ras protein can transduce the signal to effect nuclear transcription factors. (8 pts)

See page 879, Fig. 20-28
 

5. Describe a role for the mitochondria in the apoptosis pathway. (8 pts)

See page 1049, Fig. 23-50

In the membrane of the mitochondria, there is a protein dimer made of two bax proteins. These can form an ion channel that allows an influx of ions into the mitochondria. When this can happen, cytochrome c is somehow released into the cytoplasm from the mitochondria. This binds to Apaf1 and causes it to activate procaspase 9, thus beginning the caspase cascade that leads to cell death.

Also in the membrane are two molecules of the bcl2 family. This dimer is usually bound to Bad if apoptosis is to occur. However, if anti-apoptosis signals are present, Bad is phosphorylated and cannot bind. The bcl2 dimer will then inhibit formation of the bax ion channel and cytochrome c will not be released from the mitochondria.
 

6. Two tumor suppressor proteins, p53 and Rb have important roles in controlling movement through the eukaryotic cell cycle in response to DNA damage caused by ultraviolet radiation. Outline the path of events that these proteins contribute to and that leads to arrest of cell-cycle progression. (10 pts)

P53 is stabilized and its transcription is enhanced when DNA damage signals are present in the cell. It is a transcription factor that activates the transcription of p21 (CIP). P21 binds to and inhibits the cyclin/cdk complex. The complex cannot phosphorylate the RB protein. When not phosphorylated, RB remains bound to the E2F transcription factor which cannot then activate the transcription of DNA synthesis related genes. This arrest progression of the cell cycle in G1.
 

7. Describe three ways that a proto-oncogene can become an oncogene. (4 pts each)

Possibilities include:

A. Transduction by a retrovirus which causes mutation or truncation of the protooncogene. When this retrovirus infects again, the transduced oncogene causes malignancy.

B. Insertional mutagenesis. A retrovirus (not oncogenic)  inserts near a proto-oncogene. The powerful retroviral enhancers activate the transcription of the proto-oncogene abnormally creating an oncogene.

C. Chromosomal translocation. A region from one chromosome exchanges places with or attaches to a different chromosome. This causes genes on one of the chromosomes to be activated in a deregulated fashion by promoter/enhancer  sequences from the other chromosome. Or  perhaps the translocation causes a change in the proto-oncogene sequence itself as a result of the translocation.

D. Spontaneous mutation or deletion of some sequences of a proto-oncogene causing a change in the gene product’s functioning or control.

E. Gene amplification (as seen in double minutes) which cause a proto-oncogene to have multiple copies instead of the normal single copy in the genome. Thus when transcribed, the proto-oncogene becomes an oncogene since far too much gene product is made.

8. Tumor-suppressor genes are also called recessive oncogenes. Why is this? (6 pts)

Since the gene product of a tumor suppressor gene plays a protective role in inhibiting the deregulation of growth controls in the cell, both alleles of the gene must be lost before the oncogenic events occur. As long as one functional allele remains, the normal protein can be made and maintain its protective function. Thus, genetically, this resembles a recessive situation, requiring two mutations or alterations of or loss of the gene before the phenotype is seen (contribution to oncogenesis).
 

9. Draw a concept map with the term cancer as the central term. Then show at least 5 different concepts that feed into the central cancer term. (10 pts)
 

Multiple possible answers here. Consult your notes from our in-class concept mapping exercise.
 

    Problem 3 question
 

Explain why the scientists in the second paper of our problem were able to measure the levels of calcium in their cells and from that prove that the T2Rs they were testing were bitter taste receptors.

 The G protein alpha subunit they put into their cells was capable of activating phospholipase C in the membrane if it was first activated by a receptor (the T2R being tested) in the presence of a bitter tastant such as cycloheximide. When the phospholipase C was activated, it created diacylglycerol (DAG) and IP3. IP3 moved to the ER membrane in the cell and opened up calcium channels. This caused an increase of calcium level in the cell which could then be used as an indicator that the receptor T2R was responding to the bitter tastant and sending off the signal to the G protein. Thus, if calcium levels rose, the receptor was a bitter-tastant receptor. If not, the receptor was probably not a bitter-tastant receptor.
 

Graders: Problem 1: Erin Brachman
              Problem 2: Mike Bruner
              Problem 3: Trusha Vasavada
              Remainder of the exam and Problem 3 question: Dr. Schmieg