Related Experiment Video
Updated: Nov 30, 2025
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Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System
Published on: August 21, 2016
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Replisome bypass of a protein-based R-loop block by Pif1
Grant D Schauer1, Lisanne M Spenkelink2,3, Jacob S Lewis2,3
1Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523; grant.schauer@colostate.edu vanoijen@uow.edu.au odonnel@rockefeller.edu.
Summary
The accessory helicase Pif1 helps the DNA replication machinery bypass difficult barriers, such as R-loops. This study shows Pif1 removes both the protein and R-loop, ensuring genome stability.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Genome stability relies on efficient DNA replication by the replisome.
- Replication forks face obstacles that can lead to genome instability and disease.
- Accessory helicases, like Pif1, aid the replisome in overcoming replication barriers.
Purpose of the Study:
- To investigate the role of the accessory helicase Pif1 in bypassing replication barriers.
- To elucidate the mechanism by which Pif1 facilitates the bypass of inactive Cas9 (dCas9) R-loop structures.
Main Methods:
- In vitro reconstitution of the *Saccharomyces cerevisiae* replisome.
- Demonstration of Pif1's ability to bypass dCas9 R-loop barriers.
- Single-molecule fluorescence visualization to observe Pif1's mechanism.
Main Results:
- Pif1 enables the reconstituted replisome to bypass inactive dCas9 R-loop barriers.
- Bypass efficiency was similar for dCas9 R-loops on either DNA strand.
- Pif1 simultaneously removes the dCas9 protein and the R-loop during bypass.
Conclusions:
- Pif1 plays a previously unrecognized role in replication barrier bypass.
- Pif1 acts as a general displacement helicase for overcoming R-loops and protein blocks.
- This function of Pif1 is crucial for maintaining genome stability during replication.
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