Mechanism of lesion verification by the human XPD helicase in nucleotide excision repair

Abstract

In nucleotide excision repair (NER), the xeroderma pigmentosum D helicase (XPD) scans DNA searching for bulky lesions, stalls when encountering such damage to verify its presence, and allows repair to proceed. Structural studies have shown XPD bound to its single-stranded DNA substrate, but molecular and dynamic characterization of how XPD translocates on undamaged DNA and how it stalls to verify lesions remains poorly understood. Here, we have performed extensive all-atom MD simulations of human XPD bound to undamaged and damaged ssDNA, containing a mutagenic pyrimidine (6−4) pyrimidone UV photoproduct (6−4PP), near the XPD pore entrance. We characterize how XPD responds to the presence of the DNA lesion, delineating the atomistic-scale mechanism that it utilizes to discriminate between damaged and undamaged nucleotides. We identify key amino acid residues, including FeS residues R112, R196, H135, K128, Arch residues E377 and R380, and ATPase lobe 1 residues 215−221, that are involved in damage verification and show how movements of Arch and ATPase lobe 1 domains relative to the FeS domain modulate these interactions. These structural and dynamic molecular depictions of XPD helicase activity with unmodified DNA and its inhibition by the lesion elucidate how the lesion is verified by inducing XPD stalling.

A scanning-to-incision switch in TFIIH-XPG induced by DNA damage licenses nucleotide excision repair

“…Different mechanisms of DNA entry pore ordering in XPD may depend on the presence of a normal versus a damaged nucleotide ( 64 ). Hence, one way of triggering XPG by XPD may involve XPG encountering two different XPD conformations that arise from whether XPD encounters a damage or not.…”

Differing structures and dynamics of two photolesions portray verification differences by the human XPD helicase

“…The deviations of the XPD fold mainly stem from the displacement of the Arch domain with respect to the FeS domain ( Supplementary Figure S3 ). These results indicate that the XPD pore can be altered when encountering a lesion near the entry, while XPD always retains a correctly folded pore when binding to undamaged ssDNA ( 67 ), in line with the ability of XPD to discriminate damaged from undamaged nucleotides.…”

“…We have previously generated a cryo-EM-based (PBD ID 6RO4 ( 44 )) model of XPD in complex with undamaged ssDNA with an extension situated outside the entry pore of XPD ( 67 ). Importantly, this model is a translocation-capable one – it includes the incoming DNA extended outside the entry pore and it has an unoccupied space within the pore for one additional nucleotide—which permits investigation of translocation.…”

“…Importantly, this model is a translocation-capable one – it includes the incoming DNA extended outside the entry pore and it has an unoccupied space within the pore for one additional nucleotide—which permits investigation of translocation. Details concerning this model of XPD complexed with undamaged DNA are given in MATERIALS AND METHODS in our previous MD study ( 67 ).…”

“…The 6−4PP structure is based on that in the crystal structure of a damaged DNA duplex bound to Rad4–Rad23 (PDB ID 6CFI ( 68 )) and the CPD structure is based on the crystal structure of a nucleosome which contains CPD, with PDB ID 5B24 ( 69 ). The 5′ first two nucleotides (undamaged thymines) of the extended DNA (with sequence dTTTG) that were outside the pore in previous work ( 67 ), were replaced with a lesion with its bases 5T and 3T positioned outside the edge of the XPD pore. In both lesions, we retained the position of the 5T phosphate moiety as in PDB ID 6RO4 ( 39 ) and the modified bases outside the pore are oriented toward the ATPase lobe 1 helix (residues 215−221), giving a model of the XPD complexed with ssDNA containing a 6−4PP lesion ( XPD-64PP-R ) and a CPD lesion ( XPD-CPD-R ) (Figure 1C ).…”