"Helicase" Activity promoted through dynamic interactions between a ssDNA translocase and a diffusing SSB protein

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Replication protein A (RPA) is a crucial eukaryotic single-stranded DNA-binding protein involved in genome maintenance.
• RPA exhibits high-affinity binding to ssDNA and can diffuse along it, potentially disrupting short duplex DNA regions.
• Understanding RPA's dynamic interactions is key to comprehending DNA repair and replication processes.

Purpose of the Study:

• To investigate the mechanism by which the *S. cerevisiae* Pif1 protein interacts with and moves human RPA (hRPA) on single-stranded DNA (ssDNA).
• To determine if Pif1 can utilize its translocation activity to displace hRPA into duplex DNA and induce DNA unwinding.
• To elucidate the combined roles of ssDNA translocases and SSB proteins in DNA unwinding and genome maintenance.

Main Methods:

• Single-molecule total internal reflection fluorescence microscopy.
• Optical trapping combined with fluorescence approaches.
• Biochemical assays to measure Pif1 translocation and hRPA displacement.

Main Results:

• *S. cerevisiae* Pif1 uses ATP-dependent 5' to 3' translocation to push hRPA along ssDNA at significant rates.
• Pif1 can displace hRPA from ssDNA into duplex DNA, causing stable disruption of at least 9 base pairs.
• Demonstrated that Pif1's translocation activity can drive hRPA into duplex DNA, leading to unwinding.

Conclusions:

• The dynamic nature of hRPA allows for reorganization even when tightly bound to ssDNA.
• A mechanism for directional DNA unwinding is presented, involving a ssDNA translocase pushing an SSB protein.
• Processive DNA helicase function requires transient DNA melting (by hRPA) and directional ssDNA translocation (by Pif1), which can be uncoupled using separate proteins.