POGIL Cell Biology Activity 6 DNA Replication MODEL 1: "Replication Bubble" The circle is an E. coli chromosome at the beginning of DNA synthesis. The original DNA strands are called "parental strands". In this model, DNA is black and RNA is gray. Each shape represents a different enzyme. "replication fork" T I M E 1
1. Which parental strand acts as a template for synthesis of a new complementary DNA strand? - neither strand - both strands - only one strand 2. a. Label the 5' and 3' ends of the new strands in the middle panel of Model 1. b. On what end of a DNA strand are new nucleotides added? c. Draw a sketch of what a nucleotide should look like before it is added to the new strand by a polymerase: 3. a. Match each enzyme's shape with its function and fill in the column on the left. Shape: Function: Enzyme Name: Enzyme that synthesizes the largest part of the new DNA strands Enzyme that forms a phosphodiester bond without adding a new nucleotide Enzyme that synthesizes a short RNA primer Enzyme that replaces RNA nucleotides with DNA Enzyme that breaks H-bonds between strands of the parent DNA helix b. Match the following root "translations" with the enzyme names in the box below: Roots: - "lig-" is a latin prefix meaning "bind" Enzyme names: - DNA polymerase - "prim-" is a latin prefix meaning "first" - helicase - ligase - "poly-" means "many", "-mer" is "unit" - primase - "heli-" means "coil or spiral" c. Fill in each box in the Enzyme Name column in the table with one of the enzymes. One enzyme will be used twice. d. Go back and label the shapes on the model with their enzyme names. e. The enzyme that does the majority of DNA synthesis is called DNA polymerase III. The enzyme that replaces RNA nucleotides with DNA is called DNA polymerase I. Add I and III in the table above and in the model. 2
4. DNA polymerases can add nucleotides in the diagram below on the left, but cannot add nucleotides in the situation shown in the diagram on the right. (Assume in both diagrams that the bottom strand is the template for DNA synthesis). DNA polymerase can use this: but NOT this: a. Think back to transcription. Does RNA polymerase have the same restriction? b. Write out in words what RNA polymerase can do that DNA polymerase cannot, based on your answer for 'a'. 5. a. Over time, one new DNA strand at each replication fork is made in pieces, while the other one is made continuously. Explain why this has to be the case in 1-2 sentences. b. In the middel panel of Model 1, label the continuously made strand the "leading strand". Label the strand made in pieces the "lagging strand". 3
MODEL 2: This model shows one entire eukaryotic linear chromosome. The original chromosome is shown before replication. The same chromosome is shown about 30 minutes after the process of DNA synthesis has begun in each of four different cells (no enzymes are shown). The chromosomes are aligned by sequence and gray = new DNA. "original" chromosome cell # 1 2 3 4 6. Look carefully at where replication must have begun in each chromosome, then choose the statement below that BEST fits the data above. DNA synthesis can start anywhere on a chromosome. DNA synthesis starts only at one place on a chromosome. DNA synthesis starts at specific locations on a chromosome. DNA synthesis starts at every location at exactly the same time. 7. Using stars, indicate the specific locations where synthesis can start on the original chromosome. These are called "Origins of Replication" 8. a. The protein that binds to origin sequences is called an "initiator protein". Do you think that initiator proteins are expressed constitutively in cells? Why or why not? b. What is the most likely role of initiator proteins? (Circle the BEST answer) - Synthesize new DNA strands - Unwind the DNA parental strands - Join fragments of newly made DNA - Recruit (bring in) the complex of DNA replication enzymes 9. In transcription and translation, the terminator and stop codons were used (respectively) to end the processes at the correct location. Keeping in mind the goal of DNA replication, what do you think stops the process of DNA synthesis? 10. When do cells need to replicate their DNA? 4
On Your Own: 1. Watch the videos marked "Replication.mov" on our website under the "Movies" link. You may need to watch them several times to get a sense of how the replication enzymes work together at a replication fork. In the Replication.mov video: a. Which part of the DNA is phosphodiester backbone, red or yellow? b. Which part of the DNA represents the nitrogenous bases, red or yellow? c. Determine which strand is the leading strand and which is the lagging strand. d. Try to identify as many enzymes as possible in the animation. 2. A cartoon of one replication bubble is shown below. On one template strand on one fork, the newly made strands are shown. Draw in the rest for the rest of the bubble and label the 3' and 5' ends as well as the leading and lagging strands. (Arrow indicates 3' end) 3. Drawn in detail below is a region of parental DNA (within a much longer chromosome) where two replication forks are approaching each other from opposite directions. Explain what enzymes will be needed and diagram how the replication will be completed when the two forks meet. (Hint: draw a diagram to the right at a future point in time to help you!) 5
4. On planet Mandoid, DNA polymerase III adds nucleotides onto the 3 end of a growing nucleic acid just like on Earth. However, Mandoid DNA polymerase III CAN start a new DNA strand on its own (it does not need an existing 3 end to add to). 3 5 5 3 a. The diagram above shows a single replication fork of a chromosome on Mandoid. Draw and label the leading and lagging strands. Include 5 and 3 labels! b. Which of the following enzymes or molecules involved in DNA replication on Earth will ALSO be found on planet Mandoid? (Circle ALL that apply) - DNA pol I - ligase - primase - helicase 5. E. coli, a prokaryote, has a single origin of replication on its one chromosome. The E. coli chromosome is about 5 million base pairs long. Each human chromosome is on average 20 times longer. Do you think human chromosomes have a single origin or many? Why? 6