Dr. Mayor - SECTION I (10 marks) 1.

Circle the most accurate answer for each of the following questions (4 points; one correct answer per question)

Which statement does not apply to the human genome: a. Around 1-2% of the human genome code for exons b. It contains 120,000 genes encoding for proteins c. About 30% of the human genome codes for genes (including exons, introns and non coding regions) d. The human genome was first sequenced using the Sanger sequencing method (using dideoxynucleotides)
The human genome was first sequenced by shotgun sequencing.

Which enzyme does recognize and bind to ssDNA in the homologous recombination repair pathway of E. coli: a. SSB-A b. MutS/MutL complex c. DnaA d. RecB e. None of the above RecA binds to ssDNA after RecBCD unzips the DNA starting from the double-strand break. Which enzyme does cleave the DNA backbone structure to introduce a nick in the base excision repair (BER) pathway: a. DNA topoisomerase I b. AP endonuclease c. ABC excinuclease d. DNA glycosylase e. MutS/MutL complex Which statement does not apply to snRNPs (snurps): a. They mediate transesterification reactions b. They are composed of both RNA and protein c. They assemble in the splicesome complex d. They modify the single nucleotides of RiboNucleotide Particles snRNPs splice out introns from pre-mRNA, but do not modify individual nucleotides.

BIOC 303, Final Exam - April 26, 2011 2. Circle the true statement(s) for each question. More than one correct answer may apply in each question (6 points; 1.5 point each) Which statement(s) is/are true for the B-form structure of DNA: a. There are 10.5 base pairs per helical turn b. The length of a full helical turn is 68 Angstrom (a full turn is 34 A) c. The stacked bases are separated by an average of 3.4 Angstrom d. The atoms forming hydrogen bonds in the Watson and Crick bases are typically distant by 10 Angstrom (hydrogen bond distances are around 3 A) Which statement(s) is/are true for proofreading mechanism of the DNA Polymerase I: a. It requires a 5-3 exonuclease activity (3 5 exonuclease) b. It remove about 1000 nucleotides from the hemimethylated sites (1 nucleotide is removed) c. It increase the fidelity of the DNA synthesis d. It removes the mismatched nucleotide during DNA synthesis Which statement(s) is/are true for 5 capping of mRNA: a. It occurs after synthesis of the whole mRNA molecule and it is coupled to the addition of the 3 polyA tail (occurs during transcription) b. It protect the mRNA from ribonucleases c. It occurs during transcription initiation when RNA pol II inserts a 7 methylguanosine (m7G) as the first nucleotide (only methylated after capping) d. It only occurs in the mitochondria of some plant organisms (no capping in mitochondria RNA)

Which statement(s) is/are true for the Hsp70 molecular proteins: a. There are specifically involved in the folding of the H1 Histone Single Particle (H1 is not hydrophobic) b. When bound to ADP these proteins bind to misfolded proteins with higher affinity c. They bind to misfolded proteins to prevent their aggregation d. They trigger the misfolding of protein domains to favor post-translation modifications e. They are chaperone proteins that promote protein folding

BIOC 303, Final Exam - April 26, 2011 Section II - DNA Structure & Chromosome Organization (16 marks) 3. The diagram bellow shows the backbone structure of a nucleic acid molecule with the nitrogenous bases depicted as B.

a) On the diagram above, designate where the 3 and 5 ends of the nucleic acid are located (1 point) 5 is at the top, 3 is at the bottom. b) What is the main difference between the backbone structure of DNA and RNA? (1 point) DNA uses deoxyribose (2C has no hydroxyl group), RNA uses ribose sugar

c) Briefly explain why is RNA unstable in vitro in alkaline conditions? (1 point) The OH group on 2C in ribose makes the phosphodiester backbone susceptible to cleavage in alkaline conditions.

BIOC 303, Final Exam - April 26, 2011 4. The diagram below shows two bases found in DNA as they would be found in a DNA duplex with the remaining structure replaced by the two black dots.

a) Which are the two bases (designated by a and b)? (2 points) a: guanine b: cytosine

b) On the above diagram, draw a line between the atoms forming the hydrogen bond(s). (1 point) Three H bonds (between NH2 and C=O on top, N-H and N in the middle, and NH2 and C=O bottom) 5. Provide three characteristics of the four histone proteins (H2A, H2B, H3 and H4) that form the core histone complex (3 points) Hydrophilic and very basic (+ charged, Lys and Arg) to bind with DNA helix turn helix motif small proteins

BIOC 303, Final Exam - April 26, 2011 6. The laboratory of Dr. Zhao generated high-resolution maping using ChIP for the genome-wide distribution of lysine methylation of Lysine 4 and 27 of Histone H3 using next generation sequencing. The figure below shows a typical example of histone methylation pattern in a small region containing both a gene actively transcribed (STAT4) and another gene not transcribed (MYO1B) in the studied cells.

a) Briefly explain what the bars in the circle above represent? (1 point) The bars indicate the frequency of DNA fragments from that region of the gene that was bound to H3K4me2.

b) Based on the data shown in the figure above, to which region of the genome is K27 dimethylation of histone H3 associated to? (1 point) H3K27me2 is associated to MYO1B.

c) Based on the data shown, which other modification(s) may perform the same function as the mono-methylation of K27 of histone H3? (1 point) Methylation of K4 of H3 appears to have the same function.

BIOC 303, Final Exam - April 26, 2011 d) Most residues modified by post-translation modifications are located in the N-termini of the histone proteins. Briefly explain why that might be. (1 point) The N-termini contain the most basic residues (Lys, Arg) responsible for histone binding to DNA for gene expression regulation.

7. The Smc1 and Smc3 proteins associated to ATP form the cohesin complex with Scc1 and Scc3. a) When does this complex start to associate with DNA? (1 point)

Beginning of S-phase before DNA replication.

b) When does this complex dissociate from DNA? (1 point)

During separation of chromatids in anaphase.

c) Briefly explain what triggers this event? (1 point)

Anaphase promoting complex degrades securin, relieves inhibition of separase to cleave Scc1.

BIOC 303, Final Exam - April 26, 2011 Section III DNA Metabolism (25 marks) 8. Briefly explain what happens to the main DNA polymerase core complex located on the lagging strand after it finishes synthesizing an Okazaki fragment (see diagram below) during DNA replication. (3 points)

DNA polymerase core disassociates from Okazaki fragment, ditching the beta-clamp DnaG primase binds to DnaB helicase, adds new RNA primer Clamp loader puts on new beta-clamp Polymerase core binds to new beta-clamp, repeats synthesis

9. During replication initiation of DNA synthesis relies on the presence of short RNA primers that are synthesized by the primase. Briefly explain what events take place to obtain a fully replicated double strand DNA without any RNA sequence after DNA synthesis by DNA polymerase II. (3 points) RNA primer removed by DNA pol I New DNA nucleotides added by DNA pol I Nicks repaired by DNA ligase

10. In eukaryotic cells initiation of replication at the origins occurs once and only once per cell cycle. a ) Order the three events that occur at the origin prior to replication in eukaryotic cells (one of the event listed below does actually not occur) (1 point) _3__ activation of the helicase by phosphorylation _2__ association of the mcm2-7 helicase with Cdt1 near the origin DNA _1__ binding of ORC complex at the origin DNA _X__ unwinding of the DNA mediated by PCNA b) How is premature initiation of replication in G1 (that normally occurs in S phase) avoided in the cell? (2 points) Commitment to replication requires cyclin-CDK complexes to phosphorylate the pre-RC, only found in S phase Other cyclins/CDKs inhibit phosphorylation of the pre-RC complex until S phase

BIOC 303, Final Exam - April 26, 2011

11. Briefly explain what is a telomerase and what is its function in the cell. (3 points) Telomerase is a reverse transcriptase (makes DNA from RNA transcript) Telomerase adds DNA sequence repeats to the end (telomers) of chromosomes to prevent excessive shortening of telomers due to incomplete replication of the lagging strand Shortened telomers = shortened lifespan

12. Spontaneous de-amidation of cytosine converts the base to uracil. a) Which DNA repair pathway repair these small damages on bases? (1 point) Base-excision repair b) In this context, briefly explain what would be the consequence of having uracil bases on the DNA instead of thymine (1 point) Repair mechanisms would be unable to tell if uracil was correct or a result of cytosine deamidation, would increase mutations of C to U, increase A=T percentage in DNA

13. There are several events that occur during the mismatch repair pathway in prokaryotes. In which order do the following steps occur? Rank each step 1 to 5 in the space provided (1 point) __5_ DNA ligase seals the nick __2_ Scanning of the DNA to identify hemimethylated GATC sites __1_ Binding of the MutS/ MutL complex to mismatched base __3_ Cleavage of the new (unmethylated) strand by MutH __4_ Unwinding and degradation of the new DNA strand

14. Xeroderma Pigmentosum (XP) is a genetic disease, for which patients are sensitive to UV light. Some XP patients carry a mutation in the XP-G gene (which becomes inactive). XP-G is homologous to the E. coli ABC excinuclease (and share the same function). Culture of cell fibroblasts from healthy individuals and XP patients that are carrying the mutated XP-G gene are irradiated with UV light. DNA is isolated and denaturated. The resulting single strand DNA (ssDNA) is analyzed by ultra centrifugation. The ssDNA from healthy patients shows a significant reduction in the average molecular weight compare to ssDNA isolate from the treated cells carrying the XP-G mutation and from untreated cells. a) Briefly explain why there is a reduction of the ssDNA molecular weight in wilde-type cells and not in cells carrying out the XP-G mutation? (3 points) The inability to perform nucleoside-excision repair can cause base adducts to build up on the unrepaired DNA with lesions to increase its molecular weight

BIOC 303, Final Exam - April 26, 2011 b) Why would the difference not be apparent when analyzing double strand DNA instead? (1 point)

c) Culture of cells from patient carrying the mutated XP-V gene, that encodes for a TLS (translesion) Polymerase are also irradiated with UV light. Briefly explain what result would be obtained when analyzing ssDNA from these treated cells by ultra centrifugation? (1 point) M.W. would be lower because polymerases would not be able to replicate past bulky lesions

15. The error-prone translesion (TLS) polymerase contains an additional PAD domain that other DNA polymerases do not have. a) What is the main function of this PAD domain? (1 point) Stabilizes DNA binding to polymerase. b) Briefly explain why it is required? (1 point) Allows for incorporation of damaged nucleosides into larger active site.

16. During the double strand break (DSB) DNA repair by homologous recombination, the RecBCD complex processes both ends of the DNA to generate 3 overhangs. a) What are the two enzyme activities associated to RecBCD. (2 points) Helicase, exonuclease b) Briefly explain what would happen if the RecBCD generated instead a 5 overhang? (1 point) DNA polymerase would have to synthesize DNA in 3 to 5 direction, would be incomplete because of Okazaki fragments

BIOC 303, Final Exam - April 26, 2011 Section IV RNA Metabolism (9 marks) 17. What elements on DNA are recognized by the prokaryotic RNA polymerase complex to initiate transcription at specific sites in the genome? (2 points)

RNA polymerase complex binds to promoter (usually -70 to +30) with conserved consensus AT rich sequences at -10 and -35

18. Briefly explain how the lariat structure is formed during mRNA splicing to create a 2-5 phosphodiester. You can either use group II or splicesosome mediated splicing as an example. (3 points)

Group II: no enzymes or ATP involved 2 OH on adenosine in intron attacks 5 splice site to make 2-5 phosphodiester bond Splicesosome: U1 binds to 5 splice end, U2 binds to A in intron, U4 -U6 bind, kicks out U1 and U4, A acts as nucleophile to 5 19. There are several events that occur during the initiation of transcription in eukaryotes. In which order do the following events occur? (rank each step 1 to 5 in the space provided; 1 point) _1__ Binding of TATA binding protein (TBP) to TATA box _2__ Recruitment of RNA Polymerase II _3__ Unwinding of DNA near the start site _5__ Release of RNA Polymerase from the promoter region _4__ Phosphorylation of the CTD of RNA Polymerase II

20. Briefly explain what is the function of Drosha and Dicer during the processing of miRNA. (2 points) Drosha: endonuclease that cuts pri-miRNA to pre-miRNA (60-70nt) Dicer: cuts pre-miRNA to miRNA with small RNA complement

What is the role of miRNA in the cell? (1 point) For gene expression modification (usually gene silencing) by binding to DNA

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BIOC 303, Final Exam - April 26, 2011 Section V Protein Homeostasis (15 marks) 21. In the table bellow is the genetic code of the codons

Consider the following DNA coding strand: A T G G A A C G T C T AA T T A A T a) Using the genetic code table, deduce the sequence of the protein translated from this region using the first reading frame. (1 point)

Met Glu Arg Leu Ile - Asn

b) Briefly explain the consequence of a mutation that induces the substitution of the underlined guanine to a thymine? (1 point) New sequence: Met Stop Substitution causes nonsense mutation

c) Underline a region of the DNA that contains a palindromic sequence of at least 4 base pairs. (1 point) AATT or TTAA

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BIOC 303, Final Exam - April 26, 2011 22. In human cells there 50 different cytosolic tRNAs that are activated by aminoacyl tRNA synthesases. a. How many aminoacyl tRNA synthesase(s) are present in the cell? (1 point) 20, one for each amino acid. b. Briefly explain how an aminoacyl tRNA synthesase recognizes its specific amino acid? (1 point) Each aminoacyl tRNA synthetase can only bind its one specific amino acid at its amino acid arm c. Provide one reason why the aminoacyl tRNA synthesase does not recognize its specific tRNA(s) via the anti-codon region. (1 point) Some aminoacyl tRNA synthetases bind more than one mRNA codon because certain amino acids are coded for by multiple codons

23. Initiation of prokaryotic translation can be separated in three different steps. In which order do the following events occur (rank each step 1 to 3 in the provided space; one of the events does not occur)? (1 point) _X__ Recognition of the Kozak sequence _1__ Binding of IF1, IF3 and mRNA to 30S subunit _2__ Association of the 50S subunit _3__ Binding of fMet-tRNAfMet and IF2-GTP on the P site

24. What is the function of the Shine-Dalgarno sequence? (2 points) Helps guide the 30S subunit to the right place near the start AUG codon of mRNA and not other AUG codons purine-rich sequence pairs with pyrimidine rich sequence in 16s rRNA of 30S subunit

25. Why is the elongation factor EF-Tu important for the fidelity of translation (2 points) EF-Tu binds to aminoacyl-tRNA, only hydrolyzes GTP if correct codon-anticodon pairing made Increases fidelity by preventing random weak binding of tRNA to codons

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BIOC 303, Final Exam - April 26, 2011 26. Briefly describe what events occur after the peptide bond formation (see diagram below) to allow the addition of the next activated aminoacyl-tRNA during elongation of translation. (3 points)

ribosome shifts one codon to 3 end by action of EF-G, using 1 GTP deacylated tRNA moves to E site, gets released new codon is in A site, allows for binding of new aminoacyl-tRNA

27. Briefly explain what determines the tertiary structure of a protein. (1 point) Interactions such as hydrophobic packing and disulfide bonds determine tertiary structure.

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