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bioRxiv · 10.1101/2023.02.07.527508

Characterization of human XPD helicase activity with Single Molecule Magnetic Tweezers

Mao, C.·Mills, M.
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Abstract

XPD helicase is a DNA unwinding enzyme involved in multiple cellular processes. 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 (dsDNA) unwinding and single-stranded DNA (ssDNA) translocation activities of human XPD by itself. In our experimental setup, hXPD exhibits low unwinding processivity of ~14 bp and slow overall unwinding rate of ~0.3 bp/s. Individual unwinding and translocation events were composed of fast and slow runs and pauses. Analysis of these events gave similar mean run sizes and rates for unwinding and translocation, suggesting that unwinding is a reflection of translocation. The analysis also revealed that hXPD spent similar time stalling and unwinding. hXPD translocated on ssDNA at a similar overall rate as that of unwinding, pointing to an active helicase. However, we observed modest effects of DNA sequence on stalling and unwinding initiation position. Considering the slow unwinding rate, high probability of base pair separation at the ssDNA/dsDNA fork, and the observed DNA sequence dependences, we propose that hXPD is most likely a partially active helicase. Our results provide detailed information on the basal activity of hXPD which enhances our mechanistic understanding of hXPD activity. SIGNIFICANCEHuman XPD helicase is a major component of the general transcription factor TFIIH. TFIIH is essential in both transcription and nucleotide excision repair. Mutations in hXPD are associated with cancers and autosomal recessive disorders. Here we directly measured the dsDNA unwinding and ssDNA translocation of human XPD helicase by itself. Our measurements provide detailed information on the basal activity of human XPD, which enhance our mechanistic understanding of the activity of XPD in the cell, provide a basis for better understanding of the clinical phenotypes, and aid in drug design targeting hXPD related diseases.

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Mao, C., Mills, M.. 2023-02-07. Characterization of human XPD helicase activity with Single Molecule Magnetic Tweezers. https://doi.org/10.1101/2023.02.07.527508

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