MCB121 Practice problems Lecture 3 _4

MCB121 Practice problems Lecture 3 _4 - I II D NA...

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Unformatted text preview: I II D NA replication III ­1 An incorrect nucleotide incorporated into a growing DNA chain by DNA polymerase will cause the enzyme to stall before another nucleotide can be added. Describe the events that would allow the polymerase to resume DNA synthesis. III-2 The PCNA clamp has a donut-shaped structure with a center hole large enough to encircle a DNA double helix and a layer of water molecules. This protein can also interact with DNA polymerase. How might these two features contribute to increasing the processivity of polδ? III-3 DNA replication of chromosomal DNA is semi-conservative. What is the meaning of this term? III-4 DNA replication of chromosomal DNA is semidiscontinuous. What is the meaning of this term? III-5 DNA polδ has a 3' to 5' exonuclease activity. What is the purpose of this function? III-6 In 1953, a paper was published describing a model for DNA as a double helix written by Jim Watson and Francis Crick, based on X-ray data obtained by Roslin Franklin. Watson and Crick noted that the structure of DNA suggested a mechanism for its replication, yet this mechanism would present “problem” for replicating linear chromosomes: Following replication the two daughter chromatids would theoretically be shorter at one end, with the opposite ends being shorter for the two chromatids. Over time this problem would lead to the loss of DNA from the ends of chromosomes A. What aspect of the DNA structure suggested a model for DNA replication? B. What aspect of their model led them to consider that DNA would be lost from telomeres at each round of DNA replication. Draw a diagram showing how this would happen. III ­7 Draw all the nucleic acids of a bidirectional replication fork. a. Identify and label all newly synthesized strands by name. b. Show DNA segments using straight lines and the location of RNA using zig ­zag lines. c. Indicate all relevant 5’ and 3’ ends d. Indicate direction of fork movement using arrows. e. Mark the region where the origin of replication would be III ­8 Match the following term with the definition listed below. A. DNA helicase B. DNA ligase C. DNA polymerase D. DNA primase E. DNA topoisomerase F. Lagging strand G. Leading strand H. Replication fork I. RNA primer J. ssDNA binding protein (RPA) K. Sliding clamp L. Clamp loader _____ Enzyme that opens the DNA helix by separating the single strands _____ A protein complex that encircles the DNA double helix and binds to DNA polymerase, keeping it firmly bound to the DNA while it is moving _____ Y-shaped region of a replication DNA molecule at which the two daughter strands are formed _____ Enzyme that binds to DNA and reversibly breaks a phosphodiester bond in one or both strands, allowing the DNA to rotate at that point. _____ One of the two newly made strands of DNA found at a replication fork. It is made by continuous synthesis in the 5’– to - 3’ direction. _____ Short length of RNA synthesized on the lagging strand during DNA replication and subsequently removed. _____ One of the two newly made strands of DNA found at the replication fork. It is made in discontinuous segments that are later joined covalently. _____ Non enzymatic protein that prevents annealing of unwound DNA strands at the replication fork. _____ Enzyme that joins two adjacent DNA strands together III-9 Draw a diagram illustrating the addition of a dNTP to an RNA primer during DNA synthesis. You do not have to draw the structure of the bases or of the template DNA. Be sure to include the following: a. The last rNMP of the primer and the incoming dNTP. b. The chemistry of the reaction indicating the relevant reactive groups. c. The products of the reaction. III ­10 You are investigating DNA synthesis in a mammalian tissue culture line and perform the following analysis. You synchronize the cells by arresting the cells in G1 of the cell cycle (prior to DNA replication) followed by release from arrest. Thirty minutes after the release from the arrested condition you incubate cells with a thymidine analog that you can detect using immunofluorescences microscopy (shown below as solid lines). After 15 minutes you add a second thymidine analog that can be detected using a different fluorophore than the first (shown as dashed line. You carry out DNA combing analysis to better visualize the replication track. You collect images and see patterns that look like the ones shown below. A. On the diagram above, mark the likely site of all origins and replication forks. Specify the origin(s) from where each fork initiated replication. B. Indicate the relative timing of firing for each origin (e.g. “early” or “late”). What aspect of the data leads you to this conclusion? C. Describe or draw a diagram of how you would imagine the forks would be spatially organized in the nucleus. Describe the relative position of each fork relative to one another and to their corresponding origins. III ­11 Shown below is a 32P ­labeled substrate for measuring ATP ­dependent helicase activity using gel electrophoresis. The top strand was labeled by incorporation of a radiolabeled nucleotide using DNA polymerase. 5’ ** * * *3’ 3’____________|||||||||||||||||||||5’ A. This reaction will require a primer. Indicate where the primer used to label this DNA would be. B. Which of the radiolabeled nucleotides shown below would you select for this experiment? (choose one). 1. ATP, [α-32P] 2. ATP, [β-32P] 3. ATP, [γ-32P] 4. dATP, [α-32P] 5. dATP, [β-32P] 6. dATP, [γ-32P] C. Name three general properties of a DNA polymerase. D. Assuming that the helicase moves 3’-5’ relative to the ssDNA, draw a diagram of how the labeled products would migrate on the gel under different treatment conditions shown below. i.e what you would see on an autoradiogram developed after exposure of a gel. Substrate Helicase ATP Boiling + - + + + + + - + + - I II D NA replication - Key III ­1 An incorrect nucleotide incorporated into a growing DNA chain by DNA polymerase will cause the enzyme to stall before another nucleotide can be added. Describe the events that would allow the polymerase to resume DNA synthesis. If the polymerase stalls then the last nucleotide of the growing chain (in this case the incorrect nucleotide) will fall into a pocket that contains 3’ ­ to 5’ exonuclease activity. It is here that the incorporated nucleotide will be removed by hydrolysis. Once this cleaved nucleotide diffuses away, the correct nucleotide can now enter the catalytic pocket. III-2 The PCNA clamp has a donut-shaped structure with a center hole large enough to encircle a DNA double helix and a layer of water molecules. This protein can also interact with DNA polymerase. How might these two features contribute to increasing the processivity of polδ? When polδ disengages from the template, the interaction with the clamp prevents it from diffusing away. The clamp does not diffuse away since it encircles the DNA. The number of base pairs formed by a single polymerase is increased dramatically III-3 DNA replication of chromosomal DNA is semi-conservative. What is the meaning of this term? Each parental strand is used as a template to synthesize new DNA. The resulting DNA products each contain one parental strand and one newly synthesized strand. III-4 DNA replication of chromosomal DNA is semidiscontinuous. What is the meaning of this term? Replication occurs from 5' to 3', so on the 5' to 3' template strand, the replication occurs 5' to 3' in small fragments. On the 3' to 5' strand, replication occurs continuously. III-5 DNA polδ has a 3' to 5' exonuclease activity. What is the purpose of this function? Proofreading mismatched nucleotides incorportated into the growing chain by DNA polymerase III-6 In 1953, a paper was published describing a model for DNA as a double helix written by Jim Watson and Francis Crick, based on X-ray data obtained by Roslin Franklin. Watson and Crick noted that the structure of DNA suggested a mechanism for its replication, yet this mechanism would present “problem” for replicating linear chromosomes: Following replication the two daughter chromatids would theoretically be shorter at one end, with the opposite ends being shorter for the two chromatids. Over time this problem would lead to the loss of DNA from the ends of chromosomes C. What aspect of the DNA structure suggested a model for DNA replication? The structure of DNA is two antiparallel strands of DNA arranged in a double helix. The complementary base sequences allow for each strand to serve as the template to copy the information contained in of the other strand. D. What aspect of their model led them to consider that DNA would be lost from telomeres at each round of DNA replication. Draw a diagram showing how this would happen. The leading strand can continue all the way to the end of the chromosome, but the lagging strand cannot. The last RNA primer cannot be replaced by DNA because there is no upstream primer for DNA polymerase to extend. III ­7 Draw all the nucleic acids of a bidirectional replication fork. f. Identify and label all newly synthesized strands by name. g. Show DNA segments using straight lines and the location of RNA using zig ­zag lines. h. Indicate all relevant 5’ and 3’ ends i. Indicate direction of fork movement using arrows. j. Mark the region where the origin of replication would be III ­8 Match the following term with the definition listed below: A. DNA helicase G. Leading strand B. DNA ligase H. Replication fork C. DNA polymerase I. RNA primer D. DNA primase J. ssDNA binding protein (RPA) E. DNA topoisomerase K. Sliding clamp F. Lagging strand L. Clamp loader __A___ Enzyme that opens the DNA helix by separating the single strands __K___ A protein complex that encircles the DNA double helix and binds to DNA polymerase, keeping it firmly bound to the DNA while it is moving __H___ Y ­shaped region of a replication DNA molecule at which the two daughter strands are formed __E___ Enzyme that binds to DNA and reversibly breaks a phosphodiester bond in one or both strands, allowing the DNA to rotate at that point. __G___ One of the two newly made strands of DNA found at a replication fork. It is made by continuous synthesis in the 5’– to  ­ 3’ direction. __I___ Short length of RNA synthesized on the lagging strand during DNA replication and subsequently removed. __F___ One of the two newly made strands of DNA found at the replication fork. It is made in discontinuous segments that are later joined covalently. __J___ Non enzymatic protein that prevents annealing of unwound DNA strands at the replication fork. __B___ Enzyme that joins two adjacent DNA strands together III-9 Draw a diagram illustrating the addition of a dNTP to an RNA primer during DNA synthesis. You do not have to draw the structure of the bases or of the template DNA. Be sure to include the following: d. The last rNMP of the primer and the incoming dNTP. e. The chemistry of the reaction indicating the relevant reactive groups. f. The products of the reaction. III ­10 You are investigating DNA synthesis in a mammalian tissue culture line and perform the following analysis. You synchronize the cells by arresting the cells in G1 of the cell cycle (prior to DNA replication) followed by release from arrest. Thirty minutes after the release from the arrested condition you incubate cells with a thymidine analog that you can detect using immunofluorescences microscopy (shown below as solid lines). After 15 minutes you add a second thymidine analog that can be detected using a different fluorophore than the first (shown as dashed line. You carry out DNA combing analysis to better visualize the replication track. You collect images and see patterns that look like the ones shown below. fork 1 ori1 fork1 fork2 ori2 fork2 fork 3 ori3 fork 3 D. On the diagram above, mark the likely site of all origins and replication forks. Specify the origin(s) from where each fork initiated replication. E. Indicate the relative timing of firing for each origin (e.g. “early” or “late”). What aspect of the data leads you to this conclusion? Origins 1 and 2 fired soon after the addition of the labeled thymidine so they would be “early” Origin 3 fired after the concentration of labeled thymidine was reduced so it would be “late” F. Describe or draw a diagram of how you would imagine the forks would be spatially organized in the nucleus. Describe the relative position of each fork relative to one another and to their corresponding origins. Since ori1 and ori2 appear to have fired at the same time, it is possible that their respective forks would reside in the same replication factory. For the configuration shown above, the DNA at the forks 1a, 1b, 2a and 2b would all form one focus while the ori DNA would be separated. Since ori3 fires later it might be found in another replication factory. Since very little DNA replication has occurred it is likely that forks 3a and 3b and the origin will all be colocalized. III ­11 Shown below is a 32P ­labeled substrate for measuring ATP ­dependent helicase activity using gel electrophoresis. The top strand was labeled by incorporation of a radiolabeled nucleotide using DNA polymerase. 5’ ** * * *3’ 3’____________|||||||||||||||||||||5’ C. This reaction will require a primer. Indicate where the primer used to label this DNA would be. It would include sequences as the 5’ end of the top strand D. Which of the radiolabeled nucleotides shown below would you select for this experiment? (choose one). 7. ATP, [α-32P] 8. ATP, [β-32P] 9. ATP, [γ-32P] 10. dATP, [α-32P] 11. dATP, [β-32P] 12. dATP, [γ-32P] C. Name three general properties of a DNA polymerase. 1. The DNA chain grows in the 5’ to 3’ direction 2. Requires a primer to iniate 3. Requires a template strand D. Assuming that the helicase moves 3’-5’ relative to the ssDNA, draw a diagram of how the labeled products would migrate on the gel under different treatment conditions shown below. i.e what you would see on an autoradiogram developed after exposure of a gel. Substrate Helicase ATP Boiling + - + + + + + - + + - ...
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This note was uploaded on 03/16/2012 for the course MCB MCB 121 taught by Professor Gasser,burgess during the Winter '11 term at UC Davis.

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