In the following drawing the top strand is the template DNA

In the following drawing, the top strand is the template DNA, and the bottom strand shows the lagging strand prior to the action of DNA Polyerase I. Three Okazaki fragments are shown and the RNA primers (asterisks) have not yet been removed. A. Which Okazaki fragment was made first, the one on the left or the one on the right? B. Which RNA primer would be the first one to be removed by DNA polymerase l, the primer on the left or the primer on the right? For this primer to be removed by DNA polymerase l and for the gap to be filled in, is it necessary for the Okazaki fragment in the middle to have already been synthesized? Explain why. C. Let\'s consider how DNA ligase connects the left Okazaki fragment with the middle Okazaki fragment. After DNA polymerase I removes the middle RNA primer and fills in the gap with DNA, where does DNA ligase function? See the arrows on either side of the middle RNA primer. Is ligase needed at the left arrow, at the rightarrow , or both? D. When connecting two Okazaki fragments, DNA ligase needs to use NAD+ or ATP as a source of energy to catalyze this reaction. Explain why DNA ligase needs another source of energy to connect two nucleotides, but DNA polymerase needs nothing more than the incoming nucleotide and the existing DNA strand.

Solution

A. The right Okazaki fragment was made first because as each Okazaki fragment is being made in 5’ to 3’direction, the overall progress of the new DNA is moving towards the fork on the 5’ end.

B. The primer on the right will be removed first. The middle Okazaki fragment must already have been synthesized because the DNA polymerase needs a 3’ end to attach the replacement nucleotides to once it has removed the primer.

C. DNA Ligase is needed on the arrow on the right to connect the new nucleotide of DNA to the 5’ end of the DNA that already existed next to the old primer.

D. DNA polymerase uses the energy from the nucleoside triphosphate which it is attaching to the growing strand. DNA polymerase\'s bond making ability is powered by ATP, TTP, CTP, and GTP. In the case of DNA ligase, either of the cofactors splits and forms an enzyme-AMP complex. The complex binds to the nick, which must expose a 5 phosphate and 3 OH group, and makes a covalent bond in the phosphodiester chain. DNA ligase joins DNA molecules together by synthesizing phosphodiester bond between nucleotides at the ends of the two different molecules or at the two ends of a single molecule.