Enzymology – The Klenow fragment of E. coli DNA polymerase I, in the absence of free dNTPs, shows which activity?

Introduction:
The Klenow fragment is a large proteolytic fragment of E. coli DNA polymerase I that retains the polymerase and 3'→5' exonuclease domains but lacks the 5'→3' exonuclease domain. Understanding its retained activities is essential for its use in cloning, labeling, and blunt-end polishing.

Given Data / Assumptions:

• Klenow fragment lacks 5'→3' exonuclease activity.
• It still contains polymerase and 3'→5' exonuclease (proofreading) functions.
• Polymerase activity requires free dNTPs; exonuclease activity does not.

Concept / Approach:
Without available dNTP substrates, Klenow cannot extend DNA strands, but it can still remove mismatched or terminal nucleotides via its proofreading exonuclease. This property allows limited trimming or quality control of DNA ends even when synthesis cannot proceed.

Step-by-Step Solution:

Verification / Alternative check:
Biochemical assays show degradation of mismatched 3' termini by Klenow in nucleotide-free conditions, demonstrating persistent proofreading capability independent of synthesis.

Why Other Options Are Wrong:

• Endonuclease/nickase/helicase: activities not present in Klenow.
• No activity: incorrect; exonuclease remains active.

Common Pitfalls:
Confusing 5'→3' exonuclease (absent in Klenow) with 3'→5' proofreading (retained), or assuming all polymerase-associated activities require dNTPs.

Final Answer:
Exonuclease activity (3'→5' proofreading)