Displaced Strand Regulation of Facxpd Helicase Activity

Abstract

FacXPD is the archaeal homolog of yeast Rad3 and human xeroderma pigmentosum group D protein (XPD) helicase from Ferroplasma acidarmanus. This enzyme serves as a model for understanding the molecular mechanism of human Superfamily 2 helicase XPD involved in both transcription initiation and nucleotide excision repair, and for the related 5’-3’ helicases FancJ, Rtel and ChlR1 important for maintaining genomic integrity and DNA repair. We developed a single-molecule, high-resolution optical tweezers assay to decipher the mechanism by which a single XPD helicase unwinds dsDNA while translocating in the 5’-3’ direction. This assay monitors the unwinding of an 89-bp DNA hairpin substrate with single base pair resolution. Our substrate design allows us to control the length of a poly-dT ssDNA “translocation” strand (the strand to which XPD binds and along which it translocates), and a “displaced” strand (the strand displaced upon unwinding the duplex), located at the 5’ and 3’ tails of the hairpin, respectively. We found that the displaced strand interacts with XPD and that this interaction controls the helicase activity. When the 3’ tail of the substrate hairpin is substituted for dsDNA, a single XPD molecule displays repetitive “non-processive” bursts of substrate unwinding in which only ∼10-bp of the hairpin is unwound at a time. However, in the presence of a ssDNA displaced strand (of length ranging from 3 to 10 nt), we observe two types of activity: the same 10-bp non-processive mode as above, and also a “processive” mode in which the entire 89-bp hairpin is unwound. These data suggest two different binding modes for XPD resulting in non-processive or processive unwinding which are regulated by its interaction with the displaced strand. We propose a model for how the domains of XPD bind to its DNA substrate in these two modes.