Closer Look at Replisome – DNA Structure and Replication

by Georgina Cornwall, PhD

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    00:00 I think we are about ready to look at how the replisome comes together. We can think of the replisome basically as a cellular organelle in charge of replication, but the replisome is made up of all of these players. Let us see how it works. First of all, the replisome is this whole thing that you see here and it has got two main subunits. One part is the primosome involved in the priming process and the other part is the polymerase attachment clamp piece of the puzzle. Let us look at helicase. We know that he is going to be unwinding the DNA. And as helicase unwinds the DNA, closely associated we have primase laying down primers. Primers are made of RNA and we need a primer in order to create a three prime OH groups so that polymerase can come along and grab on. Because if there is no primer there, there is no three prime OH group and DNA polymerase cannot do his job. Primase is very important.

    01:12 Now let us look at how the strands get held open. As we know, helicase has been unwinding it and we have the help of gyrase to prevent supercoiling, but really those two strands want to spiral back up together. We need to hold them open single stranded. Binding proteins will bind along the DNA phosphate, sugar backbone to literally hold them open and straight so that the replisome has access to the template strands and can read them properly with its DNA polymerase III and lay down nucleotides. We have DNA gyrase.

    01:50 Now we look at other half of this apparatus containing the DNA polymerases. This apparatus is mainly a clamp loader and the clamp loader in the center has two arms out holding two DNA polymerases. What is the clamp for? Well, the clamp is there to line up the DNA template strands in the polymerase molecule and keep it flowing along the strands properly. So the clamp loader will grab these clamps and put them in place so that DNA polymerase can do its job freely. Looking at how it all comes together, we have a little bit of an issue with the lagging strand. We can certainly synthesize DNA reading the three prime to five prime directions with DNA polymerase III. We will lay down one primer and it is a straight shot in that direction, but the lagging strand has a little bit of an issue.

    02:52 Once primase lays down a primer, DNA polymerase will jump on to that primer and synthesize its nucleotides. We can see on the right-hand side DNA polymerase has done that job. As it is doing that job though the whole primosome is splitting open DNA. Helicase is doing its job. As it splits open DNA going into that direction, then we have to have a looping out of the lagging strands. So that lagging strand is going to loop out and DNA polymerase is going to have to jump back and add to the last primer. He will come along this way and it'll have a looping out as the primosome moves and then jump back and synthesize the lagging strand. In this manner, you can see that the clamp loader or the clamps will fall off and the clamp loader will get ready with a new clamp and stick DNA polymerase back onto the lagging strand. On the lagging strand, the new clamps put in place, DNA polymerase can then synthesize the next piece and we end up with the primers being removed and replaced by DNA polymerase I. DNA ligase, then comes in shortly behind this whole replisome apparatus and glues the fragments together. And so we have a continuous strand on both the leading and lagging after this whole sequence has happened. We call this process semi-discontinuous because one strand has continuous synthesis and the other strand has discontinuous synthesis.

    04:39 Synthesis on the lagging strand is somewhat cyclic in which we have this looping out, jumping back, looping out and jumping back and you can see a sort of the scheme of how it happens in this image sequence here and we have continuous synthesis of one strand and discontinuous on the other leading us on the concept of semi-discontinuous DNA replication.

    About the Lecture

    The lecture Closer Look at Replisome – DNA Structure and Replication by Georgina Cornwall, PhD is from the course Understanding Genetics.

    Included Quiz Questions

    1. A replication organelle that consists of helicase, primase, DNA polymerase III, and a clamp loader
    2. An organelle that is the powerhouse of the cell
    3. A single subunit enzyme used for ligation of Okazaki fragments
    4. A two subunit protein used to translate mRNA into an amino acid sequence
    5. The machinery that is needed to complete cell division at the end of mitosis
    1. They hold the DNA strands open.
    2. They stimulate the coiling and hydrogen bonding between parental strands.
    3. They synthesize Okazaki fragments.
    4. They relieve the stress of supercoiling.
    5. They load the β clamp on the DNA strand.
    1. Semi discontinuous
    2. Discontinuous
    3. Continuous
    4. Conservative
    5. Confirmative

    Author of lecture Closer Look at Replisome – DNA Structure and Replication

     Georgina Cornwall, PhD

    Georgina Cornwall, PhD

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    Not in details
    By Abdulrahman A. on 28. November 2017 for Closer Look at Replisome – DNA Structure and Replication

    These lectures are good , but it needs improvement it should be more in details , and the actions are not mentioned in its orders for example first of all initiation then elongation finally termination ..

    More explanation required
    By Natasha I. on 08. October 2017 for Closer Look at Replisome – DNA Structure and Replication

    This lecture and the one before are not so well explained. The clamp loader part and the process of semi discontinuous replication need more in depth explanation