Abundance of A:T base pairsName _______________ Spring 2005 Biochemistry 302 Exam 1 (90 points total) Directions: This exam has twenty-two questions/problems totaling 90 points. Check to make sure you have all six pages. Some questions have multiple parts so read each one carefully. Succinct answers are preferred but should you feel compelled to write me a short story, use the back of each page. Feel free to include drawings or cartoons in your answers as you see fit. Partial credit will be given and some questions may have more than one plausible answer. If you get stuck move on to the next question and come back to tackle more challenging questions later. You have up to two hours to complete the exam but it shouldn’t take you longer than an hour. 11a. What is the name of the base shown below (1 pt). uracil 1b. In what type of polynucleotide would this particular base normally be found (1 pt)? RNA 1c. Circle the tautomer that would be predicted to predominate at neutral pH (1 pt). keto form keto 2. RNA has a characteristic structural feature that makes it prone to degradation under alkaline (i.e. basic) conditions. Using either words or by showing a reaction mechanism, describe the chemistry underlying this phenomenon. (4 pts) Base activates the hydroxyl group linked to the 2′ carbon of ribose to facilitate nucleophilic attack on the phosphorus atom linked to the 3′ OH. Electronic rearrangement breaks the phosphodiester backbone in RNA and generates a 2′,3′ cyclic monophosphate derivative in the process. 3. Polynucleotides adopt certain types of secondary structure depending upon their chemical composition. Carefully examine the polynucleotide sequences shown below and 1) predict the helical configuration that each would adopt assuming conditions of physiologic pH, ionic strength, and water content 2) provide a biochemical rationale for your prediction. (4 pts) a. 5′-CGCGCGCGCG-3′ b. 5′-GUACCGUAGGCGACCCUACGGUAC-3′ 3′-GCGCGCGCGC-5′ Z-form A-form dsDNA with CG repeats self complementary RNA hairpin 4. DNA has the propensity to denature under conditions of high temperature or pH. Provide a chemical and thermodynamic explanation for why this is the case. (4 pts) High temperature or pH (↑ [hydroxide ion]) disrupts Watson-Crick hydrogen bonding between base pairs resulting in strand separation. Conversion from dsDNA to ssDNA is an entropically favorable process augmented by increased temperature (∆G = ∆H- T∆S). 25. Predict how and explain why the migration of a relaxed plasmid would differ from its supercoiled counterpart during non-denaturing electrophoresis? (3 pts) Because it has a more open and extended conformation, a relaxed plasmid would migrate more slowly than its more compact supercoiled counterpart. 6. In both prokaryotes and eukaryotes, zones of initiation of DNA replication exhibit a structural feature that makes them susceptible to strand separation. What is it? (2 pts) Abundance of A:T base pairs 7. For each of the functions described below, list the protein and/or enzyme of the E. coli replisome responsible on the line provided. Please use general names, which describe the activity rather than E. coli-specific gene names like DnaG or DnaB. (8 pts) a. Initiation of Okazaki fragment Primase b. Relief of superhelical stress Topoisomerase or DNA gyrase Unwinding of dsDNA template Helicase c. d. Sealing of nicks in lagging strand DNA ligase 8. What are the three different enzymatic activities present in E. coli DNA polymerase I? (3 pts) 5′→3′ polymerization, 5′→3′ exonuclease (RNA primer removal), 3′→5′ exonuclease 9. E coli DNA polymerase III holoenzyme is much more processive than single chain DNA polymerase I. A) Define what is meant by processivity in kinetic terms and in the context of DNA synthesis (2 pts). B) Describe, in structural terms, the mechanism by which the holoenzyme acquires and maintains this distinguishing characteristic (3 pts). A) Number of nucleotides incorporated in primer/daughter strand per encounter of enzyme with DNA template strand. B) Pol III holenzyme is a multi-subunit complex. Certain subassemblies enhance its processivity. Subunits comprising the γ complex (or clamp loader) facilitate the formation of a circular clamp (dimer of β subunit) around the DNA. Protein-protein interaction between core polymerase and the β clamp, which presumably can slide along the DNA, tethers the holoenzyme to the DNA (or prevents its dissociation from the DNA). 310. Selecting thymine rather uracil as the fourth base in DNA was probably an evolutionarily critical choice. Speculate why is it advantageous for a living organism to have an A, T, G, C genome rather than an A, U, G, C genome? (3 pts) Cytosine deamination, a relatively frequent occurrence, yields uracil. Such a change is mutagenic. Base excision repair system would be unable to distinguish a genomic U from one that arose due to spontaneous, non-enzymatic modification. 11. Describe the functional role of an excinuclease. (3 pts). An excinuclease cuts at two distinct sites along a DNA strand so that a specific single-stranded segment can be removed from dsDNA. 12. E. coli RecA and SSB are similar in terms of their polynucleotide binding properties but differ in their mode of action. What type of polynucleotide does each protein bind and for what biochemical purpose? (4 pts) Both interact cooperatively and with single-stranded DNA. SSB prevents reannealing of complementary strands or secondary structure formation. RecA, however, forms a helical filament to facilitate strand invasion and ATP-dependent strand exchange in a dsDNA containing a complementary sequence. 13. For each of the repair systems listed in the left column, write the letter of the enzyme or protein listed in the right column, which participates in that system. Note: There may be more than one enzyme/protein for each repair system (12 pts). I. Direct repair F A. RecA II. Base excision repair C, D, E B. DNA polymerase III III. Nucleotide excision repair D C. DNA N-glycosylase IV. Mismatch repair B, D, G D. DNA ligase V. Recombinatorial Repair A, B, D, G E. Abasic endonuclease F. Alkyltransferase G. SSB Multiple choice (2 pts each). Write the letter of the best answer on the line provided. 14.