Regulation of XPD Helicase by the Single-Stranded DNA Binding Protein RPA2

Abstract

Although it is known that single-stranded DNA binding proteins can stimulate helicase activity, the mechanism by which this occurs may be more complex than sequestering ssDNA products of duplex separation. Here, we present a single-molecule helicase assay with base-pair sensitivity, which utilizes high-resolution optical tweezers combined with microfluidics and fluorescence microscopy to decipher how FacXPD helicase is modulated by FacRPA2. FacXPD is the archaeal homolog of yeast Rad3 and human xeroderma pigmentosum group D protein (XPD) helicase from the organism Ferroplasma acidarmanus. This enzyme serves as a model for understanding the molecular mechanism of human Superfamily 2B helicase XPD involved in transcription initiation and nucleotide excision repair and related helicases FANCJ, RTEL and CHLR1 involved in maintenance of the genomic integrity. First, we demonstrated that monomeric XPD unwinds duplex DNA in single base-pair steps, yet is non-processive, unwinding for short distances (∼12 bp) and displaying a strong dependence on DNA sequence. Second, we investigated how RPA2 by itself interacts with DNA. We showed that RPA2 can unwind duplex DNA in steps of ∼5-8 bp in the presence of an assisting force of 12 pN. Finally, we examined the effect of RPA2 on XPD activity. Using our microfluidic platform, we performed experiments in which XPD and RPA2 were sequentially assembled on a DNA substrate in a controlled order. RPA2 increased XPD processivity. Duplex unwinding occurred in 1-bp steps, indicating that RPA2 is not directly involved in strand separation. Importantly, our data suggest that only 1-3 RPA2 molecules are sufficient to turn XPD into a processive helicase. We propose two scenarios. Either RPA2 forms a complex with XPD, or it alters its interaction with DNA upon binding, activating it for processive unwinding. We discuss the biological implications of our findings.