Strand-specific Recognition of DNA Damages by XPD Provides Insights into Nucleotide Excision Repair Substrate Versatility

Claudia N Buechner,Korbinian Heil,Gudrun Michels,Thomas Carell,Caroline Kisker,Ingrid Tessmer

doi:10.1074/jbc.m113.523001

Abstract

Recognition and removal of DNA damages is essential for cellular and organismal viability. Nucleotide excision repair (NER) is the sole mechanism in humans for the repair of carcinogenic UV irradiation-induced photoproducts in the DNA, such as cyclobutane pyrimidine dimers. The broad substrate versatility of NER further includes, among others, various bulky DNA adducts. It has been proposed that the 5'-3' helicase XPD (xeroderma pigmentosum group D) protein plays a decisive role in damage verification. However, despite recent advances such as the identification of a DNA-binding channel and central pore in the protein, through which the DNA is threaded, as well as a dedicated lesion recognition pocket near the pore, the exact process of target site recognition and verification in eukaryotic NER still remained elusive. Our single molecule analysis by atomic force microscopy reveals for the first time that XPD utilizes different recognition strategies to verify structurally diverse lesions. Bulky fluorescein damage is preferentially detected on the translocated strand, whereas the opposite strand preference is observed for a cyclobutane pyrimidine dimer lesion. Both states, however, lead to similar conformational changes in the resulting specific complexes, indicating a merge to a "final" verification state, which may then trigger the recruitment of further NER proteins.

Highlights

XPD is important for DNA lesion recognition by the nucleotide excision repair (NER) system
Consistent with the biolayer interferometry (BLI) results, our atomic force microscopy (AFM) analyses demonstrate that taXPD can bind to fully base paired dsDNA (Fig. 1) but binds preferentially at ssDNA regions within a DNA bubble, as well as to DNA fragment ends (Fig. 1D)
Lesion specificities of taXPDK170A obtained from AFM imaging on DNA substrates containing a DNA bubble 5Ј or 3Ј to a fluorescein or cyclobutane pyrimidine dimer (CPD) lesion (Fig. 2, striped bars) are consistent with the background level resulting from a slight preference for loading at an unpaired DNA bubble site over homoduplex DNA

Summary

Background

XPD is important for DNA lesion recognition by the nucleotide excision repair (NER) system. The crystal structure of taXPD in complex with a short stretch of ssDNA, as well as reverse footprinting analysis, have led to a model of the possible path of the DNA across the enzyme [11, 18] In this model, the DNA threads through the protein pore and is in close proximity to the iron-sulfur cluster, consistent with a proposed role of such clusters in DNA damage investigation (19 –23) and the recent identification of a dedicated lesion recognition pocket near the pore [12]. The exact knowledge of the lesion position within the DNA substrate allows us to distinguish between bound protein complexes (bound at the lesion site) and nonspecifically bound complexes (bound elsewhere on homoduplex DNA) We exploited this approach to investigate the ability of XPD to recognize and verify two different types of lesions and to directly visualize conformational responses of the complexes to damage verification. Our AFM data unambiguously show different DNA strand selectivity for the two lesions, indicating that taXPD utilizes distinct verification strategies for structurally diverse types of DNA damage

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION