Whiteboard Review Draw what Meselson and Stahl would have observed in the centrifuge tube after cycle 1 if DNA replication was ● conservative. ● semiconservative. ● dispersive. Whiteboard Review Start with “heavy” DNA. Draw the DNA strands after 4 cycles of replication with “light” DNA. DNA Replication “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” Watson & Crick (1953) Whiteboard Draw a DNA nucleotide. Hint: circle pentagon rectangle My image doesn’t have a rectangle as a base. Oh well. Whiteboard Draw a second DNA nucleotide covalently connected to your previous drawing. Whiteboard Draw a two additional DNA nucleotides hydrogen bonded to your previous drawing. Be Prepared to Answer this Question How do the bonds found in DNA fit the mechanism for copying DNA? BILL: Watch and listen to the animation. Write down all key terms related to DNA replication. How nucleotides are added in DNA replication 1. Helicase breaks hydrogen bonds between bases, unzips and unwinds the helix DANCE MOVE: Helicase breaks hydrogen bonds between complementary strands of parent DNA molecule Counts 1 and 2 Hands together move straight out from body. Represents the replication fork and the action of helicase creating two single strands at the replication fork. Whiteboard Do not erase when done... ● Draw a replication BUBBLE of DNA. ● Use an arrow to show where the origin of replication is located ● Label the 5’ and 3’ end of each DNA strand (you can decide which is which, just be sure it’s antiparallel) ● Add two helicase enzymes, one at each FORK. Usually drawn as ovals 2. Gyrase AKA “topoisomerase” Ahead of the replication fork, gyrase unwinds the DNA supercoil Whiteboard Add a gyrase to each replication fork. 3. Single Stranded Binding Proteins Hold the DNA strands apart (keeps the separated strands apart and stabilize the unwound DNA). DANCE MOVE: Single stranded binding proteins prevent the parent DNA molecules from reconnecting Counts 3 and 4 Arms move towards each other and away from each other two times. Represents the DNA strands “wanting” to reconnect, but not being able to. Whiteboard Add SSBP to each replication fork. 4. PRIMASE AND PRIMERS “Primer” DANCE MOVE: Primase adds an RNA primer onto the leading strand Count 5 Right hand with 5 fingers moves to left shoulder. The right hand represents primase placing the primer. Whiteboard Create a table comparing DNA to RNA 5. DNA Polymerase III DNA polymerase III adds DNA nucleoside triphosphates to the Huh? RNA primer sequence in a 5’ to 3’ direction. What? Ponder….. Where does energy for building DNA come from? Whiteboard Draw ATP, GTP, TTP, and CTP Hint: circles pentagon rectangles of different size Adding Bases DNA polymerase III can only add nucleotides to 3’ end of a growing DNA strand –needs a “starter” nucleotide to bond to New strand only grows 5’ → 3’ Whiteboard Draw this… Label additional 5’ and 3’ ends 3’ Whiteboard In a different color, add the new DNA strands that are being made and label their 3’ and 5’ ends. 3’ Whiteboard What is the “problem” or “limitation” with DNA polymerase III only being able to add to the 3’ end of a growing strand? Add in DNA polymerase III to show it building new DNA at the 3’ end of the new strands. 3’ Leading Strand DNA polymerase III can synthesize a complementary strand on one side of the template in the 5’ to 3’ direction with no problem DANCE MOVE: DNA polymerase III builds off the primer and moves in the same direction as helicase, towards the replication fork, building a complementary strand. It begins at the 5’ end of the new daughter strand and moves in the 3’ direction. Counts 6 and 7 Right hand on left shoulder starts with 5 fingers out (from the previous step). This represents the 5’ end of the daughter strand. Then the hand flips to two fingers out and to 3 fingers out. The fingers change from the 5 to 2 to 3 representing DNA polymerase III moving from the 5’ to 3’ end of the daugher strand (5’ “to” 3’) DANCE MOVE: DNA polymerase III moves in a continuous motion down the leading strand of the DNA template. Count 8 The right hand with 3 fingers moves along the left hand towards the wrist. At the end of the motion, your two hands should be next to each other. The left hand has 5 fingers exposed (representing the 5’ end of the parent DNA strand) and the right hand has 3 fingers expose (representing the 3’ end of the daughter DNA strand). Look! Your hands are antiparallel! What about the other strand?? Lagging Strand DNA polymerase III must work away from the replication fork. Makes a short strand of DNA, called an Okazaki fragment. As the bubble widens, it can make another short strand, and so on. DANCE MOVE: Now we’ll replicate the lagging strand. Again, the primase adds an RNA primer Count 1 The left hand with 5 fingers moves towards the elbow of the right arm DANCE MOVE: DNA polymerase III also works in a 5’ to 3’ direction on the growing lagging strand. Counts 2 and 3 The left hand on the right elbow still has 5 fingers out (from the previous step). This represents the 5’ end of the daughter strand. Then the hand flips to two fingers out and then to 3 fingers out. The fingers change from the 5 to 2 to 3 representing DNA polymerase III moving from the 5’ to the 3’ end of the daughter strand. DANCE MOVE: DNA polymerase III adds complementary DNA nucleotides to the lagging strand working away from the replication fork creating a short Okazaki fragment. Count 4 The left hand, still with three fingers out, moves towards the right shoulder (in the opposite direction as the replication fork was opening). DANCE MOVE: To create another Okazaki fragment, again the primase adds an RNA primer. Count 5 The left hand with five fingers moves towards the wrist of the right arm. DANCE MOVE: DNA polymerase III still works in a 5’ to 3’ direction on the growing lagging strand. Counts 6 and 7 The left hand on the right wrist still has 5 fingers out (from the previous step). This represents the 5’ end of the daughter strand. Then the hand flips to two fingers out and then to 3 fingers out. The fingers change from the 5 to 2 to 3 representing DNA polymerase III moving from the 5’ to the 3’ end of the daughter strand. DANCE MOVE: DNA polymerase III adds complementary DNA nucleotides to the lagging strand working away from the replication fork, creating a second short Okazaki fragment. Count 4 The left hand, still with three fingers out, moves towards the right elbow (in the opposite direction as the replication fork was opening). 6. DNA Polymerase I RNA primers are removed and replaced with DNA nucleotides by DNA Polymerase I. DANCE MOVE: DNA polymerase I removes the RNA primers from the leading strand (only 1). Counts 1 and 2 Fingers on right hand double “brush off the left shoulder (the spot where the primer was added) DANCE MOVE: DNA polymerase I removes the RNA primers from the leading strand (only 1). Counts 3, 4, 5, and 6 Fingers on left hand double “brush off the right elbow and then the right wrist (the sports where the primer as added) 7. DNA Ligase Along the lagging strand the Okazaki fragments are joined by DNA Ligase to form a single DNA strand. DANCE MOVE: Ligase seals up the breaks in the sugar phosphate backbone between the Okazaki fragments. Counts 7 and 8 and 1 and 2 Left hand circles around the right wrist, as if “taping” the wrist with a sports tape wrap. 8. Proofreading DNA polymerase I –proofreads & corrects typos –repairs mismatched bases –removes abnormal bases –reduces error rate from 1 in 10,000 to 1 in 100 million bases DANCE MOVE: Enzymes double check to make sure the replication preceded properly with little mutation. Counts 3 to 8 As if holding a book up to the nose and reading across the page, move your head from left to right between hands in front of your face. Imagine you are “proofreading.” How nucleotides are added in DNA replication 7.1.U2: reference covalent and hydrogen bonds and how the ease (or not) of breaking the bonds relates to DNA replication 7.1.U5: create a table with enzyme name and function 2.7.U2: draw a picture showing the location and function helicase 2.7.U3: explain how DNA poly III uses a template 7.1.U3: draw a picture showing DNA polymerase adding a nucleotide (circle, pentagon, rectangle) to a primer in a 5’ to 3’ direction 7.1.U4: compare replication on the leading and lagging strand
© Copyright 2024