376: Pfh1's Balancing Act: Unwinding, Rewinding, and the Role of Mitochondrial SSB

Ortiz-Rodríguez M et al., Proceedings of the National Academy of Sciences (PNAS) - Single-molecule optical tweezers and fluorescence reveal how the S. pombe Pif1-family helicase Pfh1 alternates ATP-dependent unwinding and ATP-modulated rewinding at replication-fork-like substrates, and how mitochondrial SSB spRim1 tunes those activities. Key terms: Pfh1 helicase, Pif1-family, DNA unwinding, spRim1, single-molecule.

Study Highlights:
Using single-molecule optical tweezers and fluorescence, the authors show Pfh1 performs ATP-dependent unwinding–rewinding cycles with an intrinsic ~20–22 bp processivity. Contacts with the translocating strand modulate apparent ATP affinity while engagement of the displaced strand limits maximum unwinding velocity. The mitochondrial SSB spRim1 binds the displaced strand, disrupts those contacts, and increases unwinding and rewinding velocities. Rewinding is ATP-dependent and proceeds via a sliding-back mechanism rather than strand switching.

Conclusion:
Pfh1 balances unwinding and rewinding through coordinated ATP-dependent interactions with both fork strands; binding of spRim1 to the displaced strand disrupts inhibitory helicase–strand contacts and accelerates fork dynamics, providing a mechanistic framework for how Pif1-family helicases promote replication fork progression without disrupting replisome organization.

Music:
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Article title:
Regulation of Pfh1 helicase activity by nucleic acid interactions and mitochondrial SSB

First author:
Ortiz-Rodríguez M

Journal:
Proceedings of the National Academy of Sciences (PNAS)

DOI:
10.1073/pnas.2602528123

Reference:
Ortiz-Rodríguez M, Singh SP, Cao-García FJ, Galletto R, Ibarra B. Regulation of Pfh1 helicase activity by nucleic acid interactions and mitochondrial SSB. PNAS. 2026;123(21):e2602528123. doi:10.1073/pnas.2602528123

License:
This episode is based on an open-access article published under the Creative Commons Attribution 4.0 International License (CC BY 4.0) – https://creativecommons.org/licenses/by/4.0/

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QC:
This episode was checked against the original article PDF and publication metadata for the episode release published on 2026-05-26.

QC Scope:
- article metadata and core scientific claims from the narration
- excludes analogies, intro/outro, and music
- transcript coverage: Audited the transcript sections describing Pfh1 unwinding/rewinding cycles, force and ATP dependencies, spRim1 modulation, DNA fork vs RNA–DNA fork experiments, and the proposed sliding-back mechanism and its biological relevance.
- transcript topics: Pfh1 helicase function and 5'-3' directionality; Unwinding–rewinding cycles and ~20 bp processivity; ATP concentration and force dependencies (Km(f), Vmax); Role of spRim1 in DNA fork unwinding/rewinding; RNA–DNA fork experiments and strand-switching debate; Rewinding mechanism and ATP hydrolysis effects

QC Summary:
- factual score: 10/10
- metadata score: 10/10
- supported core claims: 7
- claims flagged for review: 0
- metadata checks passed: 4
- metadata issues found: 0

Metadata Audited:
- article_doi
- article_title
- article_journal
- license

Factual Items Audited:
- PfH1 operates via unwinding–rewinding cycles with coordination between fork strands
- Intrinsic unwinding processivity is ~20 bp (not extending beyond ~22 bp per burst)
- Unwinding veloc...