Home › Analytical Chem › Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase
Analytical Chem JoVE (Open Access) Citable · DOI

Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase

DOI: 10.3791/1919-v
What you'll learn
  • ✓ Assemble a functional replisome and initiate leading strand synthesis in vitro
  • ✓ Observe replication fork stalling upon head-on RNA polymerase collision
  • ✓ Characterize Mfd-mediated replication restart mechanisms after transcription–replication conflicts
Protocol

The fate of the replisome following a collision with a head-on RNA polymerase (RNAP) is unknown. We find that the replisome stalls upon collision with a head-on RNAP, but resumes elongation after displacing the RNAP from DNA. Mfd promotes replication restart by facilitating displacement of the RNAP after the collision.

Difficulty
advanced
Total time
~4–6 hours per experiment (including protein complex preparation and real-time synthesis monitoring)
Biosafety
BSL-1

Steps

1
Prepare halted RNAP elongation complex

Generate a stalled RNA polymerase–DNA complex on a labeled DNA template. This serves as the obstacle that will collide with the replication fork.

▶ 01:19
2
Prepare downstream replication fork substrate

Construct a DNA template containing a partially replicated fork positioned to encounter the halted RNAP in a head-on configuration.

▶ 03:07
3
Assemble replisome and initiate leading strand synthesis

Load the DnaB helicase and associated replication machinery onto the fork substrate, then monitor DNA synthesis progression and collision dynamics in real time.

▶ 04:06
4
Analyze direct replication restart after collision

Record replisome stalling upon RNAP encounter, measure RNAP displacement kinetics, and quantify replication restart efficiency with and without Mfd protein.

▶ 05:49
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