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Other Replication Proteins

by Kevin Ahern, PhD
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    00:00 Now there are other replication proteins and I wanna say a few words and illustrate to you the concepts about how they work.

    00:07 If I take a DNA polymerase and I take the 4 deoxyribonucletides necessary to make DNA, and I take a strand of DNA, as I have seen right here on the screen.

    00:17 When I try to replicate that, what I discover is it will not replicate.

    00:21 Now you might say well that's the function of DNA polymerase but it turns out DNA polymerases have built into them another need. And the other need they have is that that they will only extend a strand that has already started. So you can see on this figure that the top red is a strand that has already gotten started.

    00:43 And if I put those together with this and by the way this strand that got started is called the primer, okay? And the primer is an RNA molecule not a DNA molecule.

    00:54 So the polymerase will extend from an RNA primer forwards and when I put those things together I make the molecule that you see above.

    01:04 Now as you might imagine that RNA primer on there is gonna have to be removed at some point in order for replication to be complete.

    01:12 And we will see how that is different in a prokaryotic system versus a eukaryotic system and that has some very interesting implications.

    01:20 Another molecule involved in replication is single strand binding protein.

    01:25 As its name suggest its role is binding to single strands and it turns out that single strands are pretty hazardous things.

    01:33 They are hazards is because a break in a single strands, lets every thing fly away.

    01:36 There is no information on the other strand because there is no other strand to copy and repair and replace it.

    01:43 So single strand binding protein helps maintain the integrity and hopefully protect that single strand during the time the replication is occurring.

    01:52 So we can see this occurring right here.

    01:54 If we have a replicating DNA in a cell and there is a bare single strand that usually it will have that single strand binding protein on there.

    02:03 As the replication proceeds, the single strand binding proteins will be released and go to bind other single strands.

    02:13 Now this helicase enzyme is a very interesting enzyme.

    02:17 This enzyme has the role of peeling apart the strands of a DNA duplex.

    02:26 Now that is pretty cool. This helicase has to do this so that it is not slowing down the DNA polymerase; because, the strand have to be pulled apart in order for the polymerase to replicate. Now here is the amazing thing.

    02:42 The polymerase replicates at the rate of 1000 nucleotides a second.

    02:47 A 1000 nucleotides a second is about 100 turns of the DNA per second.

    02:54 And if you multiply that out, that means that DNA has to be unwinding at the rate of 6000 rpm.

    03:01 That's faster than the engine in your car. It's really remarkable process and it's facilitated by helicase.

    03:06 Now the helicase does this seemingly without effort, although, it does use ATP in the process of making this happen.

    03:14 Well as you might imagine that if we peel things apart really quickly with the helicase, then ahead of that place we are peelings things hard what we may be doing are creating overwinding.

    03:28 So you can imagine if you pull something apart like that that has two strands ahead of place for you pulling it apart it's gonna very very tight and very very overwhelmed.

    03:39 This tension that's created by peeling apart of the strands has to be relieved; because, if it's not relieved the DNA will break, the DNA will get knotted and any of those circumstances will be very very detrimental to the cell.

    03:51 Well fortunately the cell has an enzyme called the topoisomerase that specializes in reliving tension.

    04:00 So, by relieving the tension ahead of the helicase the DNA doesn’t get overwhelmed, the replication can proceed and everything is surprisingly smooth.

    04:10 Now this does this by a couple of different mechanisms.

    04:12 They are called topoisomerase 1 that work with 1 strand at a time or the topoisomerase 2 that work with both strands at the same time.

    04:23 Now the topoisomerase 2 is called gyrase and that's the enzyme that is mostly involved in the E-coli replication system.

    04:31 Completing the DNA replication, as I said, requires the removal of RNA primers.

    04:38 So how is it that RNA primers are removed and replaced? Well this requires the action of two additional proteins and a DNA polymerase. So let's take a look at how that happens.

    04:49 Let's imagine we have a bacterial DNA. Now bacterial DNAs are circular. They are not linear like the DNAs in our cells are. Bacterial DNAs are circular.

    05:00 If the cell would to take and start replication of this circle we could imagine that it might start with an RNA primer, as seen here, and in fact would wind its way around the circle until it got back up to the top.

    05:13 And when it gets to the top, we can zoom it a little bit and see what's here, on the incoming strand on the left that's the 3 prime end of that growing strand and the place where the primer was where was the 5 prime end was, okay? The primer gets removed by an RNA-cutting enzyme.

    05:33 What is an RNA-cutting enzyme? Well in E-coli it turns out that the RNA-cutting enzyme is an activity within DNA polymerase 1.

    05:42 DNA polymerase 1 does three things. It replicates DNA It proofreads read DNA and it removes RNA primers.

    05:51 And we can see that that's already happened here. It's called a 5'-3' exonuclease.

    05:58 And it's found again in the DNA polymerase of E-Coli.

    06:00 In our cells, in eukaryotic cells, we have a separate protein that performs that function.

    06:07 But nonetheless the RNA primers have to be removed.

    06:09 The gap then is filled by DNA replications. So DNA polymerase 1 fills in that little gap and you will see there is a notch up there where the two strands aren't joined.

    06:20 The two strands have to be joined in order to finish the duplex and the joining of those is made by an enzyme called DNA ligase.

    06:29 A DNA ligase's job is to join the pieces.

    06:32 Now there is only one piece here to join but as we will see in the replication fork, there are many.


    About the Lecture

    The lecture Other Replication Proteins by Kevin Ahern, PhD is from the course DNA Replication and Repair.


    Included Quiz Questions

    1. It operates at about 6000 rpm
    2. It is found behind the DNA polymerase
    3. It assists in re-winding of the DNA after the replication fork passes
    4. It helps base pairs to form
    1. They prevent the rebinding of single-strand DNA strands to create a double-stranded form and damage to the DNA with the breakage of single strands of DNA
    2. They open up the double-stranded form of DNA to single strands
    3. They uncoil the DNA helix to make it available for replication
    4. They help in the recoiling of the newly synthesized DNA around the parental strand to recreate the duplex form
    5. They help the DNA pol during the proofreading after the completion of replication
    1. To maintain the integrity of ssDNA during replication
    2. Stabilization of the replication fork structure during replication process
    3. Proofreading of the newly synthesize DNA strand
    4. Replication of DNA in E. coli bacterium
    5. Removal of RNA primers during replication process
    1. SSB proteins— help in the synthesis of RNA primer during replication
    2. DNA pol I — 5’-3’ exonuclease activity
    3. Helicase — conversion of dsDNA into single-stranded form
    4. TopoisomeraseType 1 — relax the positively or negatively supercoiled DNA
    5. DNA gyrase — introduce negative supercoils into DNA by using ATP

    Author of lecture Other Replication Proteins

     Kevin Ahern, PhD

    Kevin Ahern, PhD


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