Investigating the Enhancement of XPD Helicase Processivity by Single-Stranded Binding Protein RPA2

Abstract

Ferroplasma acidarmanus helicase FacXPD is a Superfamily 2B helicase involved in transcription initiation and nucleotide excision repair. This archaeal protein serves as a model for understanding the molecular mechanisms of yeast Rad3 and human xeroderma pigmentosum group D protein (XPD). Previous work has shown that the unwinding of double-stranded DNA by FacXPD is enhanced by the single-stranded DNA binding protein FacRPA2. However, the mechanism by which unwinding enhancement occurs remains unknown. Here, we monitored the unwinding of a DNA hairpin by XPD in the presence of RPA2 using a single-molecule optical trap assay. By loading XPD and RPA2 onto DNA in a controlled sequence and by analyzing helicase unwinding dynamics, we distinguish between different potential mechanisms of regulation. Our data point toward a model of processivity enhancement in which RPA2 transiently interacts with the XPD-DNA complex and stabilizes a faster, more processive state of the helicase.