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 (NER). XPD uses the energy from ATP 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, XPD both unwinds dsDNA and translocates on ssDNA at the same overall rate of ∼0.3 base/s with fast runs, pauses, and backsteps within individual events. The enzyme exhibits low processivity in helicase activity, with a mean processivity of 13-16 bases unwound. The observed unwinding processivity and rate are lower than expected, suggesting that other binding partners in the cell may regulate the helicase activity of XPD. Our analysis also reveals that human XPD translocates on DNA in 1-base elementary steps. Our results provide detailed information on the basal activity of XPD which enhances our mechanistic understanding of the roles of XPD in the cell.
SIGNIFICANCE
Human XPD helicase is a major component of the general transcription factor TFIIH that is essential in both transcription and nucleotide excision repair (NER). 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 roles 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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