• Question: Thank you to everyone for your informative responses! Why is it harder for the specific ligase (I forgot its name) to attach bases in the 5' 3' direction of DNA?

    Asked by anon-244767 on 30 Apr 2020.
    • Photo: Sandra Greive

      Sandra Greive answered on 30 Apr 2020:

      Proteins that catalyse chemical reactions on nucleic acids (such as DNA or RNA) do so by binding to the nucleic acid through different kinds of specific interactions with the different components of the nucleic acid (the ribose sugars, the phosphate backbone and the nucleotide bases). The number and type of interactions between a protein and particular nucleic acid components, or groups of components, defines how specific the interaction is for each type of nucleic acid and how it is placed with respect to the protein. This then defines what part of the nucleic acid sits in the active site (the spot in the protein where the reaction happens).

      The chain of phosphoribonucleotides (nucleotides for short) that form the nucleic acid strand are joined together by a chemical reaction between the ribose rings of adjacent nucleotides. Imagine we have two nucleotides (lets call them A and B, the bases of these nucleotides could be any of the 4 usual ones for DNA: A;G; T or C). If these nucleotides are placed side by side in the same orientation, so that the 3′ position of the ribose ring of A is next to the 5′ position of the ribose ring of B. The chemical groups at these positions are held close together in the exact orientation by the enzyme so that the joining reaction (maybe ligation) can occur. The resulting chain AB starts with the 5′ position of A and ends with the 3′ position of B.

      Ligases usually join two larger nucleic acid strands together in this way by using a number of different cofactors or helper molecules.

      Polymerase enzymes add new bases to the end of the nucleic acid strand in single nucleotide steps. The nucleotide chain is held so that the 3′ position (with a hydroxyl group -OH) of the ribose ring of the last nucleotide (A) at the end of the chain is placed in the active site. This is positioned close to the incoming (new or next) nucleotide (B) which has 3 phosphate groups in a row (gamma-beta-alpha-B) attached to the 5′ position of the ribose ring. The enzyme catalyses the reaction which removes the two phosphates (the beta and gamma phosphates) furtherest away from the ribose sugar, releasing them, before joining the remaining phosphate (alpha-B) to the 3′ hydroxyl group on the ribose ring of (A-OH), nucleotide A now becomes the next to last nucleotide in the chain that ends with nucleotide B. (Note that water is released in this reaction and the phosphate group now sits between the two nucleotides to form the next position in the phosphate backbone). The polymerase moves one step along the nucleic acid so that the newly added last nucleotide (B) is in the active site and the process is then repeated to add the next nucleotide, C.

      Without knowing which enzyme you are referring to, it is hard to provide a more detailed and specific answer.

    • Photo: Alena Pance

      Alena Pance answered on 30 Apr 2020:

      This is a bit of a tricky question. Sandra explains very well why the polymerases that extend nucleotide chains (make polymers of them, hence the name) go in one direction. Ligases on the other hand generally stick two pieces of DNA together and there are two types of them. One sticks blunt ends, which means that the pieces of DNA have even ends so that there are no nuleotides sticking out of the double trand. Because of that, there is no ‘direction’ of the reaction and the pieces are glued into one randomly. The other type works on ‘sticky ends’, which means that the ends of each DNA piece form a ‘tooth’, like pieces of lego assembled in such a way to leave a protruding bit where nother piece can be ‘clicked’. The shape of the ‘teeth’ sticking out of the double strand need to be similar in the two pieces for the ligase to be able to put them together, just like the pieces of lego that have to have the same number of knobs available to put them together. In this case, the reaction is more specific.

    • Photo: Laura Devlin

      Laura Devlin answered on 30 Apr 2020:

      To add to this answer, during DNA replication, generally the DNA strand is unzipped and you have the two parent strands. One parent strand is in the 3′ – to-5′ orientation. The new copied daughter strand from this parent strand is the ‘leading strand’, as it can be made in 5′-3′, which is the direction the DNA polymerase moves.

      On the other parent strand, which is in the 5′-3′ orientation, the daughter strand will be 3′-5′, BUT the polymerase cannot move in this way, it has to move 5′-3′! So what happens? Well, the polymerase skips back and starts synthesis the daughter chain further down parent chain, moving in a 5′-3′ direction. As more of the DNA gets unzipped, the polymerase has to keep skipping back to catch up the new DNA, which is why this daughter strand is called the ‘lagging strand’. This creates lots of fragments of DNA, called Okazaki fragments, which then need to be joined together by a DNA ligase, which then covalently joins the 3’OH and 5′ phosphate group together.

      Hopes this adds a bit more information to your question.