Q.12 Which of the following activity is associated with Klenow fragment?
(A) 5′–3′ exonuclease activity
(B) 5′–3′ endonuclease activity
(C) Polymerase activity
(D) 3′–5′ endonuclease activity
Answer: (C) Polymerase activity
The Klenow fragment retains 5’→3′ polymerase activity along with 3’→5′ exonuclease activity, making option (C) correct for this CSIR NET Life Sciences question.
Option Analysis
Option (A) 5′-3′ exonuclease activity: This activity removes nucleotides from the 5′ end and is present in full DNA polymerase I but absent in the Klenow fragment due to proteolytic cleavage by subtilisin.
Option (B) 5′-3′ endonuclease activity: Endonucleases cleave internal phosphodiester bonds; neither DNA polymerase I nor Klenow fragment possesses this activity.
Option (C) Polymerase activity: Correct. Klenow fragment retains 5’→3′ polymerase activity for DNA synthesis, filling 5′ overhangs, and probe labeling.
Option (D) 3′-5′ endonuclease activity: No endonuclease activity exists; the 3’→5′ function in Klenow is exonuclease for proofreading mismatched bases from the 3′ end.
Klenow Fragment Overview
Klenow fragment derives from E. coli DNA polymerase I after subtilisin digestion, producing a large ~68 kDa fragment and a small one with 5’→3′ exonuclease. It lacks 5’→3′ exonuclease but keeps polymerase for strand extension and 3’→5′ exonuclease for error correction.
The Klenow fragment represents a key tool in molecular biology, derived from E. coli DNA polymerase I, retaining essential Klenow fragment activities like polymerase for DNA synthesis while lacking disruptive 5’→3′ exonuclease. For CSIR NET aspirants, understanding these Klenow fragment activities clarifies replication, repair, and biotech applications.
Core Klenow Fragment Activities
Klenow fragment excels in:
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5’→3′ Polymerase Activity: Synthesizes DNA from primers, fills 5′ overhangs to create blunt ends.
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3’→5′ Exonuclease Activity: Proofreads by excising mismatched 3′ nucleotides.
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Lacks 5’→3′ Exonuclease: Prevents unwanted 5′ end degradation, ideal for labeling and PCR precursors.
Comparison: Klenow vs. Full DNA Polymerase I
| Feature | Klenow Fragment | DNA Polymerase I |
|---|---|---|
| 5’→3′ Polymerase | Yes | Yes |
| 3’→5′ Exonuclease | Yes | Yes |
| 5’→3′ Exonuclease | No | Yes |
| Applications | Blunt ending, probes | Primer removal |
CSIR NET Relevance
In exams, questions test Klenow fragment activities like this MCQ, emphasizing polymerase retention over exonucleases. Variants like exo-Klenow (no 3’→5′ exo) suit labeling without degradation.
Practical Uses
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Blunt-end cloning by filling 5′ overhangs or resecting 3′ overhangs.
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Early DNA sequencing and probe synthesis before Taq dominance.
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Second-strand cDNA synthesis in biotech protocols.
