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Human PIF1 clears secondary DNA structures by coupled DNA unwinding and rewinding activities
Akshay Jayachandran1, Martin Mütze2, Kostiantyn Romaniuk1
1Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera italiana (USI), Bellinzona 6500, Switzerland.
Genes & Development
|August 10, 2026
Summary
Human PIF1 (hPIF1) helicase unwinds DNA and anneals strands simultaneously, unlike yeast Pif1 (yPif1). This DNA translocase activity clears secondary structures without extensive single-stranded DNA, maintaining genome stability.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- PIF1 family helicases are crucial for maintaining genome stability during DNA replication and repair.
- The precise biochemical activity of human PIF1 (hPIF1) is not well understood.
- Direct comparison with budding yeast Pif1 (yPif1) is needed to elucidate functional differences.
Purpose of the Study:
- To compare the biochemical activities of full-length hPIF1 and yPif1.
- To understand the distinct mechanisms of DNA unwinding and secondary structure remodeling.
- To investigate the role of hPIF1 in DNA replication fork progression and genome stability.
Main Methods:
- Bulk biochemical assays measuring DNA unwinding and annealing.
- Single-molecule magnetic tweezers experiments to analyze helicase processivity and speed.
- Mutagenesis and structural modeling to identify key functional domains.
- Assays assessing stimulation of DNA polymerase delta (Polδ)-dependent displacement loop (D-loop) extension.
Main Results:
- hPIF1 exhibits robust single-stranded DNA (ssDNA) annealing activity that limits net DNA unwinding in bulk assays.
- Single-molecule studies show hPIF1 is nearly as fast and processive as yPif1, indicating rapid reannealing, not impaired motor function, causes low net unwinding.
- Functional divergence is linked to a yeast-specific insertion in the catalytic domain.
- hPIF1 does not stimulate Polδ-dependent D-loop extension, suggesting it doesn't unwind ahead of the replication fork.
- hPIF1 efficiently remodels G-quadruplex DNA into double-stranded DNA, unlike yPif1 which produces ssDNA.
Conclusions:
- hPIF1 functions primarily as a DNA translocase, coupling local unwinding with rapid rewinding.
- This mechanism efficiently clears secondary DNA structures without generating excessive ssDNA, contributing to genome stability.
- Distinct biochemical activities of hPIF1 and yPif1 are attributed to structural differences, particularly a yeast-specific insertion.