Structural elements required for the efficient loading and activation of HELB on RPA-coated single-stranded DNA

Abstract

HELB is a human helicase involved in DNA repair and replication that interacts physically with the single-stranded DNA binding protein RPA. ATP-dependent translocation of HELB along ssDNA results in the active displacement of RPA molecules and the formation of ssDNA loops, suggesting that HELB contains at least two DNA binding sites. In this work, we investigated the role of HELB-specific structural elements in facilitating interactions between HELB and RPA-coated DNA. We show that a predicted OB-fold in the N-terminal region of the protein is important both for loop extrusion and RPA displacement. We confirm that a HELB-specific-motif within the RecA-like helicase/translocase domains is critical for binding RPA in solution but that, once HELB is bound to ssDNA, is dispensable for RPA displacement. We propose a model for RPA displacement in which both structural elements play important roles in the recruitment and activation of HELB at RPA-ssDNA filaments. SIGNIFICANCE STATEMENTSingle-stranded DNA generated during replication and repair is rapidly coated by replication protein A (RPA), creating a protected filament that must nevertheless remain accessible to DNA-processing enzymes. We show that the human DNA helicase HELB uses two specialised structural elements to overcome this problem. A HELB-specific RPA-binding motif promotes recruitment, whereas a predicted OB domain enables DNA looping and efficient RPA displacement. Removing these elements modestly enhances activity on naked ssDNA while impairing loading and activation on RPA- coated DNA. This reveals how helicase accessory domains restrain inappropriate motor activity while targeting the intended nucleoprotein substrate. Human HELB variants linked to reproductive ageing map to these regulatory regions, suggesting a connection between impaired RPA dynamics and reproductive health.