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The helicase activity of E.coli DnaB

 

Explanation:

🔬 Experiment Summary

Purpose:
To test the helicase activity of E. coli DnaB under different conditions and substrates.

🧬 Substrates Used

  1. Substrate I:

    • A partially unwound DNA (one strand radiolabeled).

    • 5′–3′ direction on the radiolabeled strand (shown in figure).

  2. Substrate II:

    • A forked DNA substrate (replication fork structure).

🧪 Experimental Conditions

ConditionDnaBATPSSBBoiling
Used to test helicase activity+++ or –+ (control)

Boiling → Denatures the DNA completely (gives fully single-stranded band = control for complete unwinding).

📊 Gel Interpretation

  • Top band → Duplex (unwound) DNA.

  • Bottom band → Single-stranded (fully unwound) DNA.

  • Boiled sample → Always gives bottom band.

Migration direction → downward (so more unwound = faster migration).

🧠 Analysis

Substrate I (partially unwound DNA):

  • DnaB + ATP → no significant unwinding (band pattern same as duplex).

  • Boiling → complete unwinding (lower band appears).

  • Adding SSB doesn’t enhance unwinding.

Substrate II (forked DNA):

  • DnaB + ATP → strong unwinding (band shifts toward single-stranded form).

  • DnaB alone or ATP alone → no unwinding.

  • DnaB + ATP + SSB → similar or slightly increased unwinding.

  • Boiling → complete unwinding (bottom band).

✅ Interpretation

  1. ATP is required for helicase activity — unwinding occurs only when ATP is present.

  2. DnaB unwinds forked DNA (substrate II) but not the blunt-ended or partially duplex substrate (substrate I).

  3. SSB (single-stranded DNA-binding protein) does not inhibit DnaB activity; may stabilize unwound DNA.

  4. Directionality: DnaB helicase acts 5′→3′ along the lagging strand template (consistent with replication fork activity).

🧾 Correct Conclusions (based on given statements)

A. “DnaB can unwind only a partially unwound DNA.” → ❌ Incorrect
→ Data show DnaB unwinds the forked substrate (substrate II), not the partially unwound duplex.

B. “SSB inhibits the unwinding activity of DnaB.” → ❌ Incorrect
→ SSB does not inhibit; activity remains similar or slightly better with SSB.

So, both A and B are incorrect.

✅ Correct Summary Statement

DnaB requires ATP and a forked DNA structure to exhibit helicase activity.
SSB does not inhibit DnaB helicase activity.


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