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In Nature (2015): Building the 21st Century Scientist UW Biology Graduates: • 47% into Health Care • 38% into Education Wednesday, January 25th, 2017 Class 14 Learning Goals DNA Replication • After this class, you should be able to: – Describe the genome-wide process of initiation of DNA replication – Define the role and predict a loss-of-function mutation result for each of the enzymes involved in replication – Given a diagram of replicating DNA, locate likely sites of action for each enzyme involved in replication – Assign descriptive terms appropriately to replication on the leading or lagging strands of a particular replication fork Polling Question #1 Which number is most likely the site of a replication origin? 1 2 A chromosome 3 4 5 6 Peer Instruction A chromosome being replicated Circular chromosomes (like in prokaryotes) Old DNA New DNA Origin of replication Linear chromosomes (like most eukaryotes) 3 5 3 5 3 What is an ‘origin’? 5 New DNA 3 5 Replication bubble Old DNA Replication fork Why do eukaryotic chromosomes have multiple origins? Why is replication able to go in both directions? Today’s activity: Each pair needs 1 packet. a few polling questions will help us to check in and keep on pace 2C: “Sketch” = basic parts (not a chemistry quiz) Polling Question #2 Which of these enzymes is labeled incorrectly? 3) Primase 4) Topoisomerase 2) DNA Polymerase III 1) Ligase Polling Question #4 Are these enzymes listed in the correct order of their operation on the lagging 5ʹ 3ʹ strand for each Okazaki fragment? 3ʹ 5ʹ 5ʹ 3ʹ Helicase, 5ʹ then primase, then DNA polymerase III, 4. DNA polymerase I removes ribonucleotides of primer, then DNA polymerase I, 1) Yes replacing them with dNTPs in the 5ʹ→ 3ʹ direction. then ligase. 2) No 5ʹ 3ʹ 5ʹ Primase synthesizes RNA primer (supplying a 3’ OH) 5ʹ 3ʹ 5ʹ Single-strand DNAbinding proteins (SSBP) Peer Instruction Topoisomerase relieves twisting forces Helicase opens double helix Why can’t DNA replication start without a primer? Why is ssBP important? What would be the phenotype of a mutation in: • Topoisomerase • Helicase Leading strand 3ʹ Peer Instruction Sliding clamp holds DNA polymerase III in place 5ʹ RNA primer 5ʹ The sliding clamp has no effect on DNA pol’s ability to catalyze one reaction. What does the sliding clamp do? Why is this called the ‘leading’ strand? DNA Replication Video In which direction is: • the replication fork moving? • the polymerase on this strand moving? Peer Instruction What is the engineering problem faced by the enzymes on the lagging strand? RNA primer 3ʹ 5ʹ 5ʹ Topoisomerase SSBPs Primase Helicase Describe the mechanisms shown here. 5ʹ 3ʹ 5ʹ Okazaki fragment Sliding clamp 3ʹ 5ʹ DNA polymerase III 5ʹ 3ʹ 3ʹ 5ʹ Okazaki fragment Okazaki fragment 5ʹ Peer Instruction Polling Question #3 Because new dNTPs can only be replicated in the 5’ to 3’ direction, we know that the following statement must be true about the strands of DNA created by DNA pol III: 1. 2. 3. 4. 5. Leading & lagging fragments are often different sizes Leading & lagging fragments are often the same size Leading moves more quickly than the lagging Lagging moves more quickly than the leading Single-stranded DNA-binding protein is not necessary Peer Instruction 5ʹ 3ʹ DNA polymerase I 3ʹ 5ʹ 5ʹ What is DNA polymerase I doing? 5ʹ 3ʹ DNA ligase 5ʹ 3ʹ 5ʹ What would be the phenotype of a deleterious mutation in the ligase-encoding gene? Lagging Strand Video Polling Question #5 Given what you know about DNA replication, which of the following do you think is most likely? 1. 2. 3. More replication errors are created on the lagging strand More replication errors are created on the leading strand Neither strand ever creates replication errors during replication Homework: Replication Enzymes Chart Enzyme ssDBP Topoisomerase Helicase DNA Polymerase I DNA Polymerase III Sliding Clamp Primase Ligase Function Location Mutation Effects? Concept Questions • How does replication begin on a single small linear chromosome? What proteins are used? – How would this be different for an extremely large circular chromosome? • Complete the given chart for the enzymes involved in replication. For each enzyme, be able to justify the evolutionary advantage and protein cost of the enzyme. • Draw an upside down ‘Y’. Assume that the tail of the ‘Y’ is double stranded DNA. Fill in the locations of all of the enzymes from the chart based on where they are likely to act. You can assume that each arm of the ‘Y’ is 500 bases long and that an Okazaki fragment is 150 bases long on average. – When finished, complete the replication bubble with the other fork. • Which strand (leading or lagging) is best characterized as: – Simple? – Likely to have mutations? – More complicated in terms of enzymes – Likely to have a very long new strand – Bonus: Slower? Peer Instruction For years, you’ve heard that genes are things that are in your DNA and can impact your life. What is a gene? 1) Does a gene include the coding region? 2) Does a gene include the non-template strand? 3) Does a gene include the stop codon? 4) Does a gene include the promoter? 5) Write a short, useful definition of a gene. 19 A working definition of the “gene” for Bio200: A gene is a unit of genetic material that encodes the information necessary to produce one useful molecule. – Usually DNA – Often encoding a protein – Not necessarily continuous – Often guided by a promoter region