Using single-molecule magnetic tweezers to study human XPD helicase activity within the core-TFIIH complex

The XPD subunit of the TFIIH protein complex is a helicase involved in the nucleotide excision repair (NER) pathway by means of creating an opening in damaged duplex DNA for other proteins to access. To form this opening, XPD hydrolyses ATP to translocate in the 5'-to-3' direction along a single strand of duplex DNA while simultaneously separating the other strand. While XPD can perform this unwinding on its own, it does so at a slow unwinding rate and with a low unwinding processivity. The structural instability of isolated XPD might be a contributing factor to this behavior. In the core complex, the XPB helicase as well as five non-helicase subunits have structural and regulatory influences on XPD activity. We used a magnetic tweezers assay to measure the double-stranded DNA unwinding activity of the core TFIIH complex, and we compared these results to previously collected XPD-only activity in identical environments. These experiments provide insight into the impact other core-TFIIH subunits have on XPD helicase activity. These results also serve as baseline data that can inform ongoing and future experiments that pair XPD with isolated TFIIH subunits, which would improve our mechanistic understanding of each subunit's individual function within the larger complex.