ATP Binding to Rad5 Initiates Replication Fork Reversal by Inducing the Unwinding of the Leading Arm and the Formation of the Holliday Junction

• Published in: Cell reports (Cambridge) Vol. 23; no. 6; pp. 1831 - 1839
• Main Authors: Shin, Soochul, Hyun, Kwangbeom, Kim, Jaehoon, Hohng, Sungchul
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 08.05.2018; Elsevier
• Subjects: Adenosine Triphosphate - metabolism; DNA Helicases - chemistry; DNA Helicases - metabolism; DNA Replication; DNA, Cruciform - metabolism; fork reversal; Models, Biological; Protein Domains; Rad5; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - metabolism; single-molecule FRET; single-molecule FRET; Rad5; fork reversal
• ISSN: 2211-1247; 2211-1247
• DOI: 10.1016/j.celrep.2018.04.029

Replication fork reversal is one of the major pathways for reactivating stalled DNA replication. Many enzymes with replication fork reversal activity have DNA-unwinding activity as well, but none of the fork reversal enzymes in the SWI/SNF family shows a separate DNA-unwinding activity, raising the question of how they initiate the remodeling process. Here, we found ATP binding to Rad5 induces the unwinding of the leading arm of the replication fork and proximally positions the leading and lagging arms. This facilitates the spontaneous remodeling of the replication fork into a four-way junction. Once the four-way junction is formed, Rad5 migrates the four-way junction at a speed of 7.1 ± 0.14 nt/s. The 3′ end anchoring of the leading arm by Rad5’s HIRAN domain is critical for both branch migration and the recovery of the three-way junction, but not for the structural transition to the four-way junction. [Display omitted] •Rad5 unwinds the leading arm using ATP-binding energy•The leading and lagging arms are proximally positioned upon ATP binding to Rad5•Rad5 migrates the four-way junction at the speed of 7.1 ± 0.14 nt/s•The HIRAN domain is critical for branch migration and replication fork recovery By using single-molecule fluorescence measurements, Shin et al. show ATP binding to Rad5 induces the unwinding of the leading arm of the replication fork, facilitating the spontaneous remodeling into a four-way junction. They show that Rad5’s HIRAN domain is critical for both branch migration and the recovery of the three-way junction.