Question

Temperature sensitive mutations are used to analyze biochemical pathways. A variety of mutations affecting DNA replication have been isolated in E. coli. What would you predict to see in the DNA after a mutation in each of the following occurred? (Answer each letter separately)

(a) DNA ligase

(b) DNA polymerase I

(c) DNA polymerase III

(d) Primase

(e) Initiator Protein

Answer 1

DNA ligase

DNA ligase is the enzyme which is responsible for joining adjacent okazaki fragments on the lagging strand. It catalyzes the formation of phosphodiester bond between those fragments.

Mutation in this enzyme will make okazaki fragments to remain separated. The lagging strand will contain separate short fragments of DNA and not a continuous fragment after replication is completed.

DNA polymerase 1

DNA polymerase 1 is responsible for removing the RNA primer present between okazaki fragments and then filling the gap between adjacent okazaki fragments.

Any mutation in this enzyme will lead to the formation of daughter DNA molecule which contains RNA primers between okazaki fragments. If incase okazaki fragments are removed, then will be present between adjacent fragments.

DNA polymerase 3

This is the enzyme which is responsible for polymerization of daughter DNA molecule. It catalyzes the formation of phosphodiester bond between two nucleotides during the polymerization process.

Any mutation in this enzyme will lead to the inability of DNA to undergo replication. This is because this enzyme is the most important of all the enzymes. It is the one which is responsible for the formation of daughter DNA strand by its polymerization activity. If this enzyme gets mutated, then polymerization and therefore DNA replication will not occur.

Primase

This enzyme is responsible for the formation of RNA primer at the starting of daughter DNA fragment. An RNA primer is required to provide free hydroxyl group at 3 prime end. DNA polymerase cannot initiate DNA polymerization on its own. It requires a free hydroxyl group at 3 Prime end. This group is is provided by primer which is synthesized by Primase enzyme.

Any mutation in this enzyme will also make parental DNA unable to copy itself. This is because even if DNA polymerase 3 is present, but there is no RNA primer, the replication will not start at all. Presence of free 3 Prime hydroxyl group is crucial to the replication process.

Initiator protein

These are the proteins that recognise origin of replication on DNA. Origin of replication is the point on DNA from where the replication starts.

Any mutation in this protein will completely halt the replication process. This is because if the starting point is not recognised properly, then all other enzymes will not be able to work.

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