Characterization of human XPD helicase activity with single-molecule magnetic tweezers

Abstract

XPD helicase is a DNA-unwinding enzyme involved in DNA repair. As part of TFIIH, XPD opens a repair bubble in DNA for access by proteins in the nucleotide excision repair pathway. XPD uses the energy from ATP hydrolysis to translocate in the 5′ to 3′ direction on one strand of duplex DNA, displacing the opposite strand in the process. We used magnetic tweezers assays to measure the double-stranded DNA unwinding and single-stranded DNA translocation activities of human XPD in isolation. In our experimental setup, hXPD exhibited low unwinding processivity of ∼14 bp and slow unwinding rate of ∼0.3 bp/s. Measurements of the ssDNA translocation activity demonstrated that hXPD translocated on ssDNA at a similar rate as unwinding, revealing that slow rate was an intrinsic property of the hXPD translocation. Individual unwinding and translocation events were composed of pauses and runs with a distribution of lengths and rates. Analysis of these events unveiled similar mean run lengths and rates for unwinding and translocation, indicating that the unwinding behavior was a direct reflection of the translocation activity. The analysis also revealed that hXPD spent similar time stalling and unwinding/translocating. The detailed basal activity of hXPD reported here provides a baseline for future studies on how hXPD activity is regulated by other TFIIH components.