Human XPD is an active helicase with low processivity and overall unwinding rate.

Abstract

XPD helicase is a DNA unwinding enzyme involved in multiple cellular processes. Of particular note is its role in the large multidomain complex transcription factor IIH (TFIIH). As part of TFIIH, XPD opens a bubble of ∼ 26 base pairs of 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 have measured the double-stranded DNA (dsDNA) unwinding and single-stranded DNA (ssDNA) translocation activities of human XPD using magnetic tweezers assays. By following changes in the extension of tethered DNA molecules under force, we are able to measure the movement of individual helicase molecule on DNA in real time. Processivity, overall rate, pause position, step size, and step rates for both dsDNA unwinding and ssDNA translocation of XPD are measured and compared. The enzyme exhibits low processivity in helicase activity, with an average ∼10 bases unwound, while the translocation of the enzyme on ssDNA is more processive, with ∼30 bases translocated. This difference in processivity on dsDNA unwinding vs ssDNA translocation may be due to interactions between the displaced strand and XPD limiting the overall processivity. However, these interactions do not affect rates, as same average rate (∼0.3 nucleotides/s) for both unwinding and ssDNA translocation has been observed. This same rate also indicates that the enzyme works as an active helicase. There is no obvious correlation between pause positions and DNA sequence. The observed low unwinding processivity (lower than the expected NER repair bubble) and rate suggests that other proteins in the TFIIH complex enhance the helicase activity of XPD.