Question

E. coli DNA polymerase III is both highly accurate and highly processive. a) Describe how Pol III avoids the incorporation of improperly paired bases. b) Describe how Pol III avoids incorporating ribonucleotides.

Answer 1

Answer/Explanation:

DNA polymerase is the enzyme responsible for replicating DNA. It is hugely important that is performs its functions accurately, as if incorrect bases are incorporated this can lead to mutations that disrupt the structure and function of genes. It adds nucleotides in a 5' - 3' direction only.

DNA polymerase III also has high processivity, which means that for every time it binds DNA, it is able to add many bases before it becomes dissociated.

A. DNA polymerase avoids the incorporation of improperly paired nucleotides in two ways:

• The first way depends on the structure of the enzyme. If the nucleotide that the enzyme is in the process of adding is not complementary to the template, then the nucleotide will not align with the template, and thus it is more inefficient to add. This inefficiency means the nucleotide is more likely to leave the active site before it is added, and DNA polymerase can replace it with the correct nucleotide.

• It also has proofreading capabilities. This means, when an incorrect base is added, it recognises the error and can fix this. It can do this because it possesses 3'-5' exonuclease activity. That means, it can chop out incorrectly added bases.

B. Ribonucleotides are the nucleotides that are incorporated into a growing RNA molecule. They are different from deoxyribonucleotides because of the differences in the sugar backbone (ribose vs deoxyribose). Their incorporation would disrupt the structure and function of the DNA, leading to problems with transcription and replication.

DNA polymerase avoids incorporating these nucleotides primarily because of the structure of the enzyme. Ribonucleotides cannot fit into the active site of DNA polymerase due to what is called a "steric filter" or "steric gate". This gate/filter function is performed by specific amino acid residues which usually have a bulky side chain and thus block the incorporation of the 2'OH of the ribose sugar (which is lacking in the deoxyribose sugar)