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Characterization of human XPD helicase activity with Single Molecule Magnetic Tweezers

Biophys J. 2023-12; 
Chunfeng Mao, Maria Mills
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CRISPR Cell Lines … Sf9 cell pellets with XPD expression were purchased from GenScript (NJ, USA). To purify XPD, … An average force calibration versus magnet position for 1 μm MyOne magnetic beads (… Get A Quote

摘要

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 (dsDNA) unwinding and single-stranded DNA (ssDNA) 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 ssDN... More

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