29. While replicating DNA, the rate of mis-incorporation by DNA polymerase is 1 in 10⁵ nucleotides. However, the actual error rate in the replicated DNA is 1 in 10⁹nucleotides incorporated. This is achieved mainly due

to

(1) spontaneousexcision of mis-incorporated

Nucleotides
(2) 3’à5′ proofreading activity of DNA polymerase

(3) termination of DNA polymerase at mis-incorporated sites

(4) 5’à3’ proof reading activity

Introduction

DNA replication is an essential cellular process that must be highly accurate to maintain genetic integrity. Although DNA polymerases incorporate nucleotides with high specificity, the initial error rate during nucleotide incorporation is approximately 1 in 10^5. However, the actual mutation rate in replicated DNA is much lower, about 1 in 10^9 nucleotides. This dramatic improvement in fidelity is primarily due to the proofreading activity of DNA polymerase.

What Causes the Discrepancy Between Initial and Actual Error Rates?

• Initial misincorporation rate: DNA polymerase occasionally incorporates incorrect nucleotides during DNA synthesis, roughly 1 error per 100,000 nucleotides.

• Final error rate: After proofreading and repair, the error rate drops to about 1 error per 1 billion nucleotides, ensuring genome stability.

This difference is mainly attributed to the proofreading function of DNA polymerase, which corrects errors immediately after they occur.

The Proofreading Mechanism: 3′→5′ Exonuclease Activity

• DNA polymerases possess a 3′→5′ exonuclease activity, which scans the newly added nucleotide at the 3′ end of the growing DNA strand.

• If a mispaired or incorrect nucleotide is detected, the polymerase pauses and transfers the DNA strand from the polymerase active site to the exonuclease active site.

• The exonuclease excises the incorrect nucleotide by cleaving the phosphodiester bond, removing the mismatch.

• The corrected primer terminus is then repositioned back to the polymerase site for continued DNA synthesis.

This proofreading step significantly reduces the incorporation of errors into the genome.

Why Other Options Are Not Responsible for the High Fidelity

1. Spontaneous excision of mis-incorporated nucleotides:

   • This is not a major contributor. DNA polymerase actively removes errors rather than relying on spontaneous removal.

2. Termination of DNA polymerase at mis-incorporated sites:

   • DNA polymerase does not terminate synthesis upon errors; instead, it corrects them via exonuclease activity.

3. 5′→3′ proofreading activity:

   • DNA polymerases do not have 5′→3′ proofreading exonuclease activity. The 5′→3′ exonuclease activity is involved in primer removal, not error correction.

Summary Table

Conclusion

The remarkable accuracy of DNA replication is largely due to the 3′→5′ exonuclease proofreading activity of DNA polymerase. This function allows the enzyme to detect and excise incorrectly incorporated nucleotides immediately, reducing the error rate from 1 in 10^5 to 1 in 10^9 nucleotides. This proofreading mechanism is essential for maintaining the genetic stability of all living organisms.

Correct answer:
(2) 3′→5′ proofreading activity of DNA polymerase