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VALENCIA CHM 1025C - AL Exercise #3 DNA Replication

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Name: DNA Replication Page 1 of 4 DNA Replication ActivityBackgroundYou are working on a new antibiotic. You suspect that this antibiotic targets the prokaryotic replication machinery. To test this, you set up a replication experiment. You add all the components needed for replication (proteins and ribonucleic ac-ids), a plasmid with all the necessary replication sequences, and radioactively labeled deoxyribonucleotides (dNTPs) to two test tubes, and then add your drug to the experimental tube, but not the control tube. Because the only dNTPs available for replication are radioactively labeled, all new DNA generated in your replication experiment will be radioactive (hot). You hypothesize that if your drug blocks replication, you will not see any hot plasmid DNA in your experimental tube. Part 1To distinguish your original plasmid from products of new replication, you decide to add nucleotides labeled with radioac-tive phosphate to the mix. Refer to the schematic below. Can you use nucleotides that are labeled with P32 at the γ location (γ-P32)? At the α location (α-P32 )? Both? Why or why not?Part 2Please draw out the plasmids you would expect to obtain after one round and after two rounds of replication (starting with a single plasmid). What do you expect in your control tube (normal replication) and in your tube with the drug if it fully blocks replication? Use a red pen for hot and a blue pen for cold strands of DNA. (Assume that when no new DNA is synthesized, the two strands of the original plasmid re-anneal back together.)Control test tube Test tube + drugStart First round Second round Start First round Second roundCheck with your instructor to make sure your answer above is correct!Name: DNA Replication Page 2 of 4Part 3You run your experiment and look at the DNA replicated after two rounds. You separate plasmid DNA from the smaller dNTPs and measure radioactivity. To your disappointment, you get hot plasmid in the test tube with your drug. You show the results to your professor, who tells you that it’s still possible that the antibiotic targets DNA replication machinery.To figure out what may be happening, draw what you would expect to see in the first round of replication. Use the schematic on the left to work out what would happen during replication. Use thin lines for RNA and thick red lines for DNA. Please pay attention to the leading and lagging strands. Note that the 5ʹ and 3ʹ here refer to the template strand directionality. Next, fill out the schematic on the right, drawing what you expect to see after the first round of replication is completed. Use thick red lines for ‘hot’ DNA (and blue for ‘cold’ DNA). (Assume that if no new DNA is generated, the two strands of the original plasmid will re-anneal back together.) The yellow bars represent the origin of replication. 1. Control reaction → 2. What would happen if you blocked the action of PRIMASE, so that it could no longer synthesize RNA? → 3. What would happen if you blocked the action of DNA POLYMERASE III, so that it could not accomplish 5ʹ → 3ʹ polymerization? →Name: DNA Replication Page 3 of 4 4. What would happen if you blocked the action of DNA POLYMERASE I, so that it could not accomplish 5ʹ → 3ʹ exonuclease reactions? → 5. What would happen if you blocked the action of DNA LIGASE? → Is it possible that your antibiotic is targeting a component of the DNA machinery? If yes, which one(s)? Explain your answer. How would the action of the antibiotic affect replication?Name: DNA Replication Page 4 of 4End-of-Activity Questions 1. What kind of replication occurs in our genetic systems and builds a new strand on a template strand? a. conservative replication b. semiconservative replication c. dispersive replication d. convergent replication 2. What happens to the phosphate group of the incoming dNTP before it is added to the growing DNA molecule? a. The OH group is cleaved off. b. The entire phosphate group is cleaved off. c. The sugar is cleaved off. d. Two phosphates are cleaved off. 3. In a replication experiment using a cold template plasmid and hot dNTPs, what would you expect to see after two rounds of replication? a. All plasmids will have two hot strands. b. All plasmids will have one hot and one cold strand. c. Half the plasmids will have two hot strands, and half will have one cold and one hot strand. d. One quarter of the plasmids will have all cold strands, one quarter will have all hot, and one quarter will have one cold and one hot. 4. What would you expect to see after one round of replication if your antibiotic blocks the 5ʹ → 3ʹ exonuclease activity of DNA polymerase I? (Select all that apply.) a. hot daughter plasmids b. no hot daughter plasmids c. no gaps in DNA nucleotide sequence on the newly synthesized strand d. gaps in DNA nucleotide sequence on the newly synthesized strand where primer RNA nucleotides are not removed e. gaps in DNA nucleotide sequence on the newly synthesized strand where primer RNA is removed but no new DNA is


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