1 Biochemistry 302, February 11, 2004 Exam 1 (100 points) Name I. Structural recognition (very short answer, 2 points each) 1. What form of DNA is shown on this Nature Genetics cover? Z-DNA or left-handed DNA 2. Name the bases (full names, not just single letters) that make-up this mismatched base-pair. guanine thymine 3. This modified base is typically found in CpG islands in eukaryotes. What is it? 5-methylcytosine 4. During what molecular process would one expect to find DNA in this configuration? DNA recombination
2 II. Short Answer (3 points each) 1. Why is radiolabeled thymidine the best substrate to use in experiments designed to determine the rate of cell proliferation in vitro? Thymidine will be incorporated into replicating DNA but not RNA. 2. Provide a biochemical rationale for why AT-rich sequences are commonly found in zones of initiation of DNA replication and near sites of transcriptional termination. Only two hydrogen bonds needed for base-pairing between adenine and thymine versus three for guanine and cytosine. As such, AT or AU-rich sequences are more susceptible to strand separation or melting. 3. What is the difference between an exonuclease and an endonuclease. An endonuclease cleaves phosphodiester bonds at sites within a polynucleotide chain while exonucleases remove nucleotides from either the 5 or 3 end. 4. How is the active site of RNA polymerase biochemically converted to that of a ribonuclease? Binding of accessory factors (such as GreA, GreB, TFIIS) donate additional amino acid residues and a metal ion to the active site. 5. List the type of DNA damage created by cisplatinin and the repair system(s) that are able to fix such lesions (complete spelling please, no abbreviations). Inter- and intrastrand crosslinks via guanine bases, nucleotide excision repair or recombinatorial repair. III. T/F (2 points each) 1. F The ability of DNA to adopt a Z-form configuration is completely independent of nucleotide sequence. 2. F DNA is positively charged at ph 7. 3. T 3 -deoxyadenosine is an inhibitor of RNA synthesis. 4. F Acetylation of histone proteins enhances their DNA-binding affinity. 5. F RNA primer excision during DNA replication is catalyzed by the ε subunit of the DNA polymerase III holoenzyme. 6. F Loading of the DNA polymerase III sliding clamp (β subunit) onto the lagging strand need only occur ONCE during DNA replication. 7. T Chemical agents that chelate divalent metal ions inhibit the polynucleotide synthetic activities of DNA and RNA polymerases.
3 IV. Multiple choice (3 points each): 1. A TFIIIA is a general transcription factor responsible for promoter recognition and transcriptional initiation of genes encoding: A. 5S rrna B. trna C. 45S rrna E. A and B only 2. F Conversion of uridylate to thymidylate requires: A. Ribosylation B. Methylation C. Reduction D. Oxidation E. Both A and D F. Both B and C 3. B The principal thermodynamic parameter that accounts for the overall stability of the DNA double helix is: A. Entropy B. Enthalpy C. Temperature D. ph E. Ionic strength 4. D Core histone proteins comprising the nucleosome possess which of the following biochemical properties: A. High arginine content B. Dimerization ability C. Substrate for acetylation E. None of the above 5. A Nitrate-induced deamination of cytosine produces which of the following mutagenic bases in DNA? A. Uracil B. Thymine C. Hypoxanthine D. 5-azacytosine
4 6. B The following activity is absent in all core eukaryotic DNA polymerases: A. 3 -exonuclease B. 5 -exonuclease C. leading strand polynucleotide synthesis D. lagging strand polynucleotide synthesis 7. A Which of the following proteins is principally responsible for stabilization of template strands in a non-base-paired conformation during DNA replication? A. SSB B. DNA Helicase C. Primase D. Topoisomerase 8. C One of the activities below is NOT required for base excision repair. A. DNA N-glycosylase B. Abasic endonuclease C. DNA helicase D. DNA polymerase E. DNA ligase 9. C The first line of defense in correction of a mismatched base-pair that is formed during the course of DNA replication in E. coli is: A. Base excision repair B. Nucleotide excision repair C. 3 -exonuclease activity of DNA polymerase I D. 5 -exonuclease activity of DNA polymerase I 10. A DNA would be predicted to be in an A-form configuration under conditions of: A. Low water content B. Absence of protein C. High temperature D. None of the above 11. D RecA exhibits all of the following activities EXCEPT: A. Single-stranded DNA-binding affinity B. Strand exchange C. ATPase D. Helicase
5 12. B Which of the following enzymes is inhibited by low concentrations of α-amanitin? A. RNA polymerase I B. RNA polymerase II C. RNA polymerase III 13. A DNA polymerase, DNA ligase, and RNA polymerase are related because each enzyme: A. Catalyzes 3-5 phosphodiester bond formation. B. Uses nucleoside triphosphate as substrates. C. Is expressed at a limiting concentration in cells. D. Requires two metal ions at the active site. 14. D TATA-binding protein (TBP) is best described as a: A. DNA-bending protein B. Initiation factor for eukaryotic transcription C. Recruitment factor for other components of the general transcription machinery E. None of the above 15. A Mediator proteins such as Rad54 assist Rad51 (the eukaryotic homolog of RecA) in initiating homologous recombination/strand exchange by: A. Enhancing recruitment of Rad51 to single-stranded DNA B. Sequestering RPA (SSB) via protein-protein interaction C. Activating the ATPase activity of Rad51 E. None of the above 16. B Depurination of DNA is induced by: A. High ph B. Low ph C. High ionic strength D. Low ionic strength 17. B Mismatch repair in E. coli is dependent on the following DNA modification: A. Purine alkylation B. Adenine methylation C. Guanine oxidation D. Depyrimidination
6 V. Structural biology (12 points) The proteins, 8-oxo-guanine DNA-N-glycosylase (OGG) and integration host factor (IHF), are related by their ability to distort the structure of DNA but each protein does so in a different way. Briefly describe/list 1) the biological function of each protein 2) the type of DNA alteration induced or stabilized by each protein, and 3) how each protein mediates the structural change in DNA at the molecular level (I m looking for a biochemical explanation here). OGG 1) Base excision repair enzyme or removes 8-oxoG from DNA by glycosidic bond cleavage 2) Flipping 8-oxoG out of the DNA helix (to facilitate catalysis) 3) Replacement of flipped 8-oxoG with an arginine residue to hydrogen bond with the estranged cytosine, van der Waals forces and H-bonding between 8-oxoG and other active site amino acids keep 8-oxoG in its flipped out state (in position for catalytic attack) IHF 1) initiation factor in DNA replication and/or site-specific recombination 2) site-specific DNA-bending 3) disruption of base-stacking by intercalation of amino acid residues (proline) between certain base-pairs in the DNA minor groove