DNA Polymerase

by Kevin Ahern, PhD

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    00:00 Well let's think a little bit about this. Let's talk first about the DNA polymerase.

    00:06 Now a DNA polymerase is an enzyme that catalyzes the formation of the strand and you can see that shown schematically at the bottom of this figure.

    00:16 You can see that the DNA polymerase is sitting on top of the strand at the end of a strand which is labelled 5 prime end. So it's sitting on the 3 prime end and you see that the direction of replication is to the right.

    00:27 All DNA replication, no exceptions, occurs in the 5 prime to 3 prime direction.

    00:35 As I said, it reads the base on one strands and then it knows what to put into the strand that it's building.

    00:39 So for example, if the DNA polymerase sees a G in the opposite strand, it will incorporate a C in the strand that it's building; because, C is complimentary to the G.

    00:51 The DNA polymerase selects the proper bases by knowledge of the base pairing rules which say the G goes with C and A goes with T for example.

    01:00 So the role of the DNA polymerase is to grab the new base, as I said, and join it in a phosphodiester bond in the growing chain.

    01:10 Now the DNA polymerase is an interesting enzyme in that many DNA polymerases especially ones found in cells has another catalytic activity associated with it called proofreading.

    01:23 Now proofreading is, you can think of like an editor.

    01:27 DNA polymerase is really good at finding the right base across from the opposite strand.

    01:32 But you have to remember that the DNA polymerase is working at an incredible rate.

    01:36 Some DNA polymerases are incorporating the growth of a strand by a 1000 nucleotides a second.

    01:44 So proofreading and checking for errors is very important; because, this DNA molecule that's made will become the genetic information for the next cell and you wanna have as few of errors as possible.

    01:53 The 3 prime to 5 prime exonuclease is an activity of DNA polymerase that checks for errors.

    01:59 So what it does is it reads every base pair not only before it puts it in but also after it has put it in and it checks for correctness.

    02:07 Now this is actually a little complicated but in simple terms what happens is that if there is a bulge that it is at the wrong base was put in, it will bulge. It won't fit the same ways as if the proper base is put in.

    02:19 So if the polymerase senses a bulge it knows it has made an error and then it backs up.

    02:25 So it does this with a 3 prime to 5 prime exonuclease, as you can see in this schematic here, and that literally is a backing up and a chewing out. It is literally taking out what it put in.

    02:37 And if that happens then the correct base can then be put in by advancing in the proper direction.

    02:45 Now proofreading we know improves the fidelity that is the accuracy with which the DNA is copied by a 100-fold.

    02:53 That's a pretty remarkable again as a result of proofreading, especially, for a process that is going as fast as DNA replication.

    02:59 Now DNA polymerases have an interesting structure and you can see the structure here.

    03:03 This is actually the ribbons part of this is actually the DNA polymerase.

    03:08 And the stick figure in the middle, it is little harder to see is the DNA being replicated.

    03:14 So I have drawn in green here a figure to show that what the DNA polymerase has is something that looks like a hand.

    03:24 And in the middle of the hand we have the DNA strand actually being held.

    03:29 Now that hand is common to many many DNA polymerases and you can see that the DNA, as I said, being held here.

    03:37 Here is a DNA polymerase known as DNA polymerase 1.

    03:39 And you can see the different regions of it that are marked here.

    03:42 Now the one on the left is a modified DNA polymerase 1 in which the proofreading part of it is being retained but the primer removal part, that I will talk about later, has not.

    03:53 I show this figure to show you again the claw that you can see and you can see the claw in the figure on the right and that's the place where the DNA molecule is held and where the synthesis actually is occurring.

    04:06 Now E-coli cells have 3 DNA polymerases that are labelled as 1, 2 and 3.

    04:15 Now many cells have multiple DNA polymerases and each polymerase tend to have its own function. There are quite a few DNA polymerases in human cells for example.

    04:25 In E-coli, the polymerase known as polymerase 3 replicates most of the genome.

    04:31 The polymerase 1 that just I showed you replicates fairly short fragments and the polymerase 2 doesn't have a major role in E-coli but it’s thought it may play some roles in repair the damage of the DNA.

    04:45 Now polymerases differ in their fidelity and their processivity. So let me explain those terms.

    04:52 Fidelity is, again, the accuracy with which the polymerase is copying the DNA.

    04:57 This is affected by the 3 prime to 5 prime exonuclease or the proofreading function.

    05:04 Not all DNA polymerases have a proofreading function but most cellular ones do.

    05:09 Viral DNA polymerases frequently lack a 3 prime to 5 prime exonuclease and that means that they are much more prone to making errors.

    05:20 And making errors for a virus is actually a pretty good thing; because, it helps it to evolve faster and that's why we see difficulty with viruses becoming resistant to drugs sometimes for example.

    05:31 Processivity is another important concept to get a hold off.

    05:34 Processivity is a description of how long a DNA polymerase will get on to a DNA and stick with it as its replicating.

    05:45 Now that might seem a little odd to think about. But in fact, there is some big differences in the way that DNA polymerases do this.

    05:52 DNA polymerase 3, the one that replicates an E-coli, is highly processive.

    05:58 It gets on to a DNA molecule and will stay on and replicating for thousands or millions of nucleotides. Whereas DNA polymerase 1 is not highly processive.

    06:09 It goes on and it comes off. It goes on and it comes off.

    06:12 Well how does it do this? DNA polymerase 3 gets onto and stays on the DNA; because, it uses a protein that you see in the figure here on the right called the clamp.

    06:24 And the clamp, not surprisingly, helps it to hold on to the DNA even better than the claw of the hand that I showed you, and it makes a little ring around it as you can see like this.

    06:32 Well with the DNA in the middle there is no place for the polymerase to go flying off.

    06:37 Polymerase 3 uses a clamp called a beta clamp.

    06:43 Polymerase 1 does not use a clamp. So polymerase 1 will go on to a DNA molecule, it will replicate and then it will fall off.

    06:50 And it will go on to another DNA molecule and it will replicate and then fall off.

    06:52 Now as we will see that that falling off is built into the types of replication that polymerase 1 is performing and the staying on is built into the types of replication that polymerase 3 is performing.

    07:08 Now, as I said, there are some viruses that have DNA polymerases that don't copy things very accurately.

    07:14 Retroviruses are very a good example. HIV is a virus that makes a lot of mutations very quickly.

    07:24 And it does that because it doesn't copy the nucleic acid very accurately.

    07:30 They have a DNA polymerase that's called a reverse transcriptase and reverse transcriptase copies RNA and makes DNA. It's one of those exceptions to the central dogma.

    07:40 The other thing associated with that DNA polymerase is odd is that it has low fidelity; because, it has no proofreading.

    07:49 And the lack of proofreading allows the retrovirus to wildly change and so that's why maintaining HIV with a drug regime can be a problem; because, the virus has built into ways of evolving itself very quickly through mutation.

    About the Lecture

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

    Included Quiz Questions

    1. It comes from the 3’ to 5’ exonuclease activity of some DNA polymerases
    2. It involves changes in the parent DNA strand
    3. It is present on all DNA polymerases
    4. It is due to a 5’ to 3’ exonuclease activity of a DNA polymerase
    1. It works mostly on the leading strand
    2. It has three enzymatic activities
    3. It does proofreading
    4. It has a hand-like structure
    1. Increases when the enzyme remains on the DNA molecule for a longer period of time
    2. Refers to how fast it completes a process
    3. Increases when the enzyme moves to different strands rapidly
    4. Is destabilized by the beta clamp
    1. It has high fidelity
    2. It is a DNA polymerase in retroviruses
    3. It uses RNA as a template to make DNA
    4. It uses deoxyribonucleotides

    Author of lecture DNA Polymerase

     Kevin Ahern, PhD

    Kevin Ahern, PhD

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