The Effect of Single-Stranded DNA Binding Protein RPA2 on XPD Helicase Processivity

Abstract

FacXPD helicase is the archaeal homolog of yeast Rad3 and human xeroderma pigmentosum group D protein (XPD) 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. Previous work has shown that the unwinding of double-stranded DNA by FacXPD is regulated by the single-stranded DNA binding protein FacRPA2. However, the mechanism by which this occurs is unknown. Here, we present a single molecule study of this regulation using optical traps. We show that XPD is a weak helicase as a monomer, only able to unwind short distances (∼12 bp) under tension applied by the optical traps, with a strong dependence on DNA sequence. In the presence of RPA2, however, XPD monomers are able to unwind more processively (>90 bp). Using a combination of optical traps and a laminar flow cell, we performed experiments in which we loaded XPD, RPA2, and provide ATP in a controlled sequence. We used this approach to distinguish between different potential mechanisms of regulation. Our data disfavor mechanisms by which RPA2 regulates XPD activity by sequestering ssDNA. Instead our results suggest that RPA2 forms a complex with XPD, activating it for processive unwinding. RPA2 can also bind ahead of the traveling helicase and destabilize the duplex junction.