Single-Molecule Analysis of Conformational Transitions in XPD Helicase

Abstract

Modular organization of DNA helicases is crucial for their cellular functions. ATP binding and hydrolysis governs sequential conformational transitions within the conserved motor core of a helicase, allowing it to transform the chemical energy stored in ATP into mechanical work or directional motion on the DNA lattice. Auxiliary modular domains incorporated into the helicase core determine substrate specificity and often modulate activity of the helicase. XPD (Xeroderma pigmentosum complementation group D) is a DNA helicase with a role in nucleotide excision repair and transcription. It is also a model for understanding the fundamental molecular mechanisms of a family of iron-sulfur (Fe-S) cluster containing helicases, which includes FANCJ, RTEL1, and CHLR1. These enzymes contain two characteristic auxiliary domains, Fe-S and ARCH, incorporated into the motor core. Previous structural and biochemical studies suggested that these auxiliary domains are involved in XPD-DNA interaction and may undergo significant conformational rearrangements during the helicase cycle. Such rearrangements will likely result in spatial separation of the Fe-S and ARCH domains. Here, we addressed whether XPD auxiliary domains are indeed dynamic and whether their mobility is correlated with DNA and ATP binding by the helicase. Single, fluorescently labeled XPD molecules were tethered to the surface and monitored using total internal reflection fluorescence microscopy. Conformational transitions of XPD were detected by following Fe-S-mediated quenching of a fluorescent dye that was site-specifically positioned in the ARCH domain. Dual excitation with green and red lasers was used to correlate conformational dynamics of XPD helicase with binding of the DNA substrate. This approach allowed us to separate and analyze the conformational dynamics of XPD in absence and in presence of DNA.