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Lecture 14

Lecture 14

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Biological Sciences
Dan Riggs

Lecture 14 – DNA replication II: Enzymology, Origins, Organization and Licensing SPs: Figs 13-12, 13, 14, 15, 19, 20 Vocabulary: helicase/primase/primosome/replisome/Sliding clamp/origin/fiber autoradiography/licensing Work by Kornberg (1950’s) -interested in DNA polymerase and how it works, how many subunits it had, what it did, etc. -he purified the protein, he took the dna template, added the raw materials for replication to occur (i.e. deoxynucleotide triphosphates dNTP’s) and he radioactively one type of the nucleotides. When the radioactive nucleotide is incorporated into the DNA polymer then the polymer will also become radioactive. This is evidence that DNA Replication is occurring and radioactive materials are being incorporated into the chromosome. -Figure 13-7: Kornberg was able to purify a variety of different templates like double stranded circles, like single stranded circles, like double stranded DNA with partial single stranded character. -He discovered that there are two goals for DNA replication: 1. DNA polymerase needs a 3’ hydroxyl group to get started at a prime site and 2. DNA polymerase has to have a template in front of it to copy. This is why, in figure 13-7, the templates on the right are better templates than the ones on the left. Enzymology at the Replication Fork -What has to go on in order for replication to take place… - Firstly, some enzyme activities must be assigned. Starting at the origin of replication, currently the DNA is double stranded and it has to be single stranded in order for DNA polymerase to read to template so one of the first things that needs to be done is that the double helix needs to be unwound by the enzyme helicase. -Now, to keep the single stranded pieces from snapping back together by complementary base pairing, single stranded DNA binding proteins exist and they coat the strands so that they can’t snap back together. -DNA polymerase can only start at a primed site, so in order to initiate strands, an enzyme called primase is needed. It lays down a short RNA primer and provides the 3’ hydroxyl group. -Now the DNA with an open end and primed site is ready for DNA polymerase to do the job. So DNA Polymerase is responsible for chain elongation from the primed site (in ecoli, there are 3 DNA polymerase types and DNA polymerase III is the main replication enzyme). -Once DNA polymeraseIII is done its job, the RNA primers that still exist need to be removed and replaced with DNA. This is done by DNA polymerase I which removes the ribonucleotides and replaces them with deoxyribonucleotides. -On the “discontinuing” or “lagging” strand, DNA is synthesized discontinuously as short Okazaki fragments. These need to be put together so to enzyme called DNA ligase is responsible for sealing that small section and creating the intact polymer chain. Enzymes at the Replication Fork: Primase Figure 13-12: -Double stranded DNA is seen on the left which is the parental strand that needs to be unwound -In the middle, is the replication fork where replication is going to occur from right to left as the DNA unwinds. -DNA helicase unwinds the DNA (it is a hexamer of 6 identical subunits). Closely associated with DNA helicase is the enzyme Primase. Primase and Helicase move along together as unwinding takes place. Primase lays down the RNA primers (green). The space is about 9- 10 nucleotides in length. The complex of Helicase and Primase is known as Primosome. -When you see double stranded molecules, it is important to know which way is which. So always put in the 5’ and 3’. Replicons, eyes, bubbles and forks -A unit of replication is referred to as a replicon. -In the context of double stranded DNA where there is an origin that exists, there is unwinding by helicase, then there is bidirectional replication. -Looking at this through a microscope, you begin to see the helix becomes unwound and you get something that looks like an eye or a bubble - it is wider than it was before. -As elongation continues, the eye or bubble gets larger. Each eye is about bidirectional replications so there are two replication forks. -The fork is where the replicated DNA is meeting the unreplicated parental DNA. Each eye consists of two forks that are moving in opposite directions. The leading and lagging strands are continuous and discontinuous so each fork then also consist of one leading strand going 5’ to 3’ continuously and one lagging strand that is synthesized as the short Okazaki pieces in a discontinuous fashion. Slide a figure 13 – 9 Here we see a little bit of a problem. There is a replication fork, partially replicated DNA on the left and a unreplicated DNA in the right. The fork is moving from the left to the right and is going to unwind. There is a rule that all DNA polymerize is have to obey - they must replicate the leading and the lagging strand in a 5’ to 3’ direction. Since the helices are anti- parallel, one has to be going to the left and the other has to go to the right to obey this rule. What you see is that the actually move physically in the same direction. How is this possible? The rules are that these two molecules of DNA polymerase III act to polymerize DNA in a 5’ to 3’ manner but they move in concert in one direction. The secret of this is looping one of the strands. If you loop the lagging strand template 180° so that it is facing the opposite direction then it's 5’ end is the same polarity as the leading strand 5’ end. See blackboard animations. Drawing Here we have a replication fork. The un replicated parental DNA is on the right and the replication is going on at the fork. The lagging strand template has been looped 180°. The direction that the loop is going in is from left to right and that means that the leading strand is going from 5’ to 3’ and the molecule of polymerase III can just keep zipping along as the parental DNA is unwound. This is the easy part. The difficult part (which has become easier since we have looped the lagging strand template) is that another molecule of polymerase III acting in concert with the first molecule operating on the leading strand is now synthesizing the lagging strand in the same direction. It is obeying the rules going 5’ to 3’ but it can move in conjunction with the other molecule of polymerase III because the lagging strand template is looped 180 degrees. The book shows this in figure 13 – 13 in a more complex fashion. Essentially, the first image is the same as what we have just seen. On the right, there is the unreplicated parental DNA and the leading strand template being copied by one molecule of DNA polymerase III, moving from left to right (moving 5' to 3'). The lagging strand template has been looped 180° and the second molecule of DNA polymerase III is finishing up an Okazaki fragment displacing the single-stranded binding protein as it moves along. The second panel shows that once it has run into this RNA primer from the last Okazaki fragment, it is going to let go of the looped template. It has completed the Okazaki and more DNA is reeled through here and looped and then the polymerize re-engages at a pre-existing primer and continues on its job to synthesize this Okazaki fragment. This is known as the trombone model. The Trombone Model: The loop changes in size as the tube is being played and you can see from the animations on blackboard how this takes place. See the animation from the McGraw-Hill textbook which was shown in lecture. There are four videos but ignore the video on the Hershey and Chase experiment. Two molecules of DNA polymerase III act together going in the same direction to replicate both the leading and lagging strand. In figure 13 – 14, shows what is going on at the replication fork. You see two molecules of DNA polymerize III as the round entities. Holding onto them is something called a Beta clamp. This is a very important thing. DNA polymerase has to do two things: First they need to hold onto DNA very tightly such that they can read the template and zip along at thousands of nucleotides per second. Second, they have to hold onto it loosely enough so that they can process the template in a very quick fashion. The Beta clamp is a multiple subunit protein which is attached to the DNA polymerase and it essentially looks like a clamp surrounding the template molecules. The job of the beta clamp is that it allows DNA polymerase to hold on to the template molecule and not lose its grip. It also allows the core polymerase to read the template, put in the complimentary nucleotide and continue on its way without dissociating from the template. Both molecules of polymerize have the clamp and they are attached together by a protein complex that consists of subunits called Tau. Tau is a multifunctional complex which holds onto both molecules of DNA polymerase. At the front end (at the replication fork) the Tao is making contact with the helicase and at the back end it makes contact with a clamp loader. When it is appropriate for the complex to let go of the lagging strand template, and then come back and latch onto it again at a new Okazaki fragment, that clamp loader spreads the clamp apart so it can attach to the new template, ATP hydrolysis occurs, the clamp closes and allows the polymerize to hold on to the template by means of the clamp. By moving the lagging strand template 180°, those two molecules can move in the same direction and still obey the rules that polymerization can only occur in a 5’ to 3’ fashion. Letting go of the clamp, reestablishing the clamp, and the replication of Okazaki fragments. In Figure 13 – 15, a lot of the main players have been taken away for simplicity. At the top is the parental DNA, on the left is the leading strand template and on the right is the lagging strand template. There is a molecule of DNA polymerase III that is engaged on the lagging strand template, started from the RNA primer in green and has now elongated the chain by incorporating DNA nucleotides shown in red. When it runs into the next Okazaki fragment, it let go of the clamp and, because the template is looped, it will recycle back to the pre- assembled site where a new primer has formed. As the helix is opened up, primase acts to put the primer down, the clamp is laid down at that place, polymerize comes back because the
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