Name _____________________________ BIO362 EXAM I Feb.

25, 2011

1.a) The nucleic acid shown below is a partially completed Okazaki fragment containing both RNA and a small number of deoxyribonucleotides. If it is hydrolyzed with alkali, what will the products be? You should answer using shorthand notation such as pA, Ap, Appp, pUpA, etc. Of course not all the products will have A as the base; they are just shown as examples of the kind of notation to use. Put the letter d in front of any deoxyribonucleotides or DNA fragments. (17 pts total for parts a and b)

1. b) Draw the complete structure of one of the products shown above, as it would be in solution at pH=7.

2. Name the reactants and products of the arginase reaction shown below. (6 pts)

3. Captain Kirk and Mr. Spock are transported to an alternate universe. Bacteria that inhabit the alternate planet earth do not express nitrogenase enzymes. How would this affect the biosynthesis of amino acids by organisms on this planet and which amino acids would be affected? (5 pts)

4. Why does serine deficiency reduce the biosynthesis of deoxythymidine triphosphate? (6 pts)

5. What is a nucleosome and how does it compact DNA? (8 pts) Nucleosome A Nucleosome is the basic unit of DNA packaging in eukaryotes, consisting of a segment of DNA wound in sequence around eight histone protein cores.[1] This structure is often compared to thread wrapped around a spool.[2] Nucleosomes form the fundamental repeating units of eukaryotic chromatin,[3] which is used to pack the large eukaryotic genomes into the nucleus while still ensuring appropriate access to it (in mammalian cells approximately 2 m of linear DNA have to be packed into a nucleus of roughly 10 m diameter). 2

Nucleosomes are folded through a series of successively higher order structures to eventually form a chromosome; this both compacts DNA and creates an added layer of regulatory control, which ensures correct gene expression. Nucleosomes are thought to carry epigenetically inherited information in the form of covalent modifications of their core histones. The nucleosome hypothesis was proposed by Don and Ada Olins in 1974[4] and Roger Kornberg.[5][6] The nucleosome core particle consists of approximately 147[7] base pairs of DNA wrapped in 1.67 lefthanded superhelical turns around a histone octamer consisting of 2 copies each of the core histones H2A, H2B, H3, and H4.[8] Core particles are connected by stretches of "linker DNA", which can be up to about 80 bp long. Technically, a nucleosome is defined as the core particle plus one of these linker regions; however the word is often synonymous with the core particle.[9] Linker histones such as H1 and its isoforms are involved in chromatin compaction and sit at the base of the nucleosome near the DNA entry and exit binding to the linker region of the DNA.[10] Non-condensed nucleosomes without the linker histone resemble "beads on a string of DNA" under an electron microscope.[11] In contrast to most eukaryotic cells, mature sperm cells largely use protamines to package their genomic DNA, most likely to achieve an even higher packaging ratio.[12] Histone equivalents and a simplified chromatin structure have also been found in Archea,[13] proving that eukaryotes are not the only organisms that use nucleosomes.

6. Putrescine is an organic compound that is largely responsible for the foul odor of putrefying flesh. Putrescine serves as a substrate for the enzyme PMT. The reaction catalyzed by PMT is inhibited in cells that are deficient in ATP. However, PMT does not directly bind and utilize ATP. Based upon this information, fill in the reactant and product in the boxes below and briefly (1-2 sentences) explain why you chose them. (8 pts)

7. Carbamoyl phosphate synthetase deficiency is an inherited disorder. Patients with this disorder are missing the enzyme carbamoyl phosphate synthetase. These individuals possess elevated levels of glutamine. Briefly explain why this is so. (8 pts)

8. Speculate on why primase does not have a proofreading exonuclease. (6 pts)

9. List one similarity between telomerase and primase and one difference. (10 pts) 1. DNA Polymerase III Responsible for DNA Replication Polymerase III is really an aggregate of ten different polypeptides and is referred to as a holoenzyme. The assembled aggregate has a total molecular weight of 760 kDa. The subunits are present in the holoenzyme in equal ratios. There are only 10-15 holoenzyme molecules per cell, and when the holoenzyme binds to the DNA, it remains firmly attached and proceeds along the growing chain until the strand is complete. The holoenzyme is a dimmer with two active sites, one for synthesizing the 4

leading strand and one for extending the lagging strand. How the holoenzyme by passes the previously synthesized strand to get the next primer on the lagging strand is not exactly understood, but it likely involves folding the DNA at the site of synthesis. 2. The Primosome is Responsible for DNA strand initiation. The primosome is a huge protein composed of 15 subunits responsible for the initiation of each new Okazaki fragment. As mentioned earlier, DNA polymerase can not initiate new strand synthesis, but RNA polymerases can initiate strand synthesis de novo. The primosome accomplishes initiation of strand synthesis by synthesizing a short segment of RNA for the DNA polymerase to use as a primer. The primase component of the primosome is responsible for synthesis of the short RNA fragment. However, it can not act alone; it must be present in the primosome complex. 3. Telomerase It adds specific DNA sequence repeats ("TTAGGG" in all vertebrates) to the 3' end of DNA strands in the telomere regions, which are found at the ends of eukaryotic chromosomes. This enzyme is active only in germ cells and cancer cells.Embryonic stem cells express telomerase, which allows them to divide repeatedly and form the individual. In adults, telomerase is highly expressed in cells that need to divide regularly (e.g., in the immune system), whereas most somatic cells express it only at very low levels in a cell-cycle dependent manner.

10. Name the amino acid on all topoisomerase that is transiently covalently attached to a DNA phosphate during the enzymes reaction cycle. (4 pts)

11. Suppose the melting temperature of DNA is compared in two solvents, 0.1 M NaCl versus a 50:50 mixture of 0.1 M NaCl and methanol. Explain why the Tm is significantly lower in the solvent containing methanol. (Hint: remember the main source of stability of the DNA double helix.) (8 pts)

12. Suppose the DNA molecule shown below represents the left end of a linear eukaryotic chromosome. (14 pts) a. Draw a replication fork that started far away from what is depicted and is moving leftward toward the end of the molecule. Draw two Okazaki fragments at the fork and indicate an RNA primer with a wiggly line on only one of them. Indicate polarities on all strands. b. After you have drawn the fork, in a separate drawing, draw the two completed DNA molecules that result when the fork reaches the left end. Assume the cell has no telomerase and this linear molecule did not have any telomeric sequence at its end. ___________________________ ________________________________________ 3'

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