25. Type of proofreading activity bv DNA polymerase I in E.coli is

(1) 5’-3’ exonuclease
(2) 5′-3′ endonuclease

(3) 3′-5′ exonuclease
(4) 3′-5′ endonuclease

Introduction

DNA Polymerase I (Pol I) in Escherichia coli is a multifunctional enzyme essential for DNA replication and repair. One of its critical roles is to maintain the accuracy of DNA synthesis through a proofreading mechanism. This proofreading activity is carried out by a specific exonuclease function that removes incorrectly incorporated nucleotides, thus preventing mutations and ensuring genomic stability.

What Is Proofreading in DNA Replication?

Proofreading is a quality control process during DNA synthesis where DNA polymerases detect and correct errors immediately after nucleotide incorporation. When a wrong nucleotide is added, the enzyme pauses and excises the mispaired nucleotide before continuing synthesis. This significantly reduces the mutation rate during replication.

DNA Polymerase I and Its Enzymatic Activities

E. coli DNA Polymerase I is unique because it possesses multiple enzymatic activities within a single polypeptide:

• 5′→3′ DNA polymerase activity: Adds nucleotides to the growing DNA strand.

• 3′→5′ exonuclease activity: Proofreading function that removes misincorporated nucleotides from the 3′ end.

• 5′→3′ exonuclease activity: Removes RNA primers ahead of the polymerase during Okazaki fragment processing and participates in DNA repair.

Among these, the 3′→5′ exonuclease activity is responsible for proofreading.

The Proofreading Mechanism of DNA Polymerase I

• During DNA synthesis, if an incorrect nucleotide is incorporated, the DNA polymerase detects the mismatch due to improper base pairing.

• The enzyme then shifts the DNA from the polymerase active site to the 3′→5′ exonuclease site.

• The exonuclease activity excises the incorrect nucleotide from the 3′ end of the primer strand.

• After removal, the DNA strand returns to the polymerase site, and correct nucleotide incorporation resumes.

This process enhances replication fidelity by approximately 100- to 1000-fold.

Why 3′→5′ Exonuclease Activity Is Crucial

• It prevents permanent mutations by correcting errors in real-time.

• It maintains the integrity of the genetic code during DNA replication and repair.

• It enables DNA Polymerase I to function efficiently in filling gaps during Okazaki fragment maturation without introducing errors.

Distinguishing Proofreading from Other Activities

Summary Table: Proofreading Activity of DNA Polymerase I

Conclusion

The proofreading activity of E. coli DNA Polymerase I is mediated by its 3′→5′ exonuclease function, which excises incorrectly incorporated nucleotides from the 3′ end of the growing DNA strand. This activity is essential for maintaining high fidelity during DNA replication and repair, ensuring the genetic information is accurately transmitted.