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* This work was supported by the Deutsche Forschungsgemeinschaft Rudolf Virchow Center for Experimental Biomedicine Grants FZ 82 (to I. T. and C. K.) and KI-562/2 (to C. K.). This article contains supplemental text, references, and Figs. S1–S6.
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.
Maintenance of genomic integrity is one of the most important cellular tasks and is largely achieved by a number of different DNA repair systems targeting diverse types of DNA lesions, such as erroneous alterations in the genetic code, chemical base modifications, or bulky adducts (
is an essential DNA repair mechanism with an exceptionally large range of chemically and structurally unrelated targets. In humans, it is furthermore the only repair system for the removal of UV irradiation-induced damages, and dysfunctional NER is responsible for severe diseases including xeroderma pigmentosum (
). Eukaryotic NER encompasses a total of ∼30 proteins, including the xeroderma pigmentosum group proteins (XPA–XPG). In the current model of NER, repair can either be initiated by a stalled RNA polymerase in transcription coupled NER or via global genome NER through high affinity binding of the XPC-HR23B heterotrimer to short distorted and destabilized DNA structures containing ss/dsDNA junctions (
). Once the damage has been verified, the NER cascade proceeds with the recruitment of additional proteins including the endonucleases XPG and XPF-ERCC1, resulting in the excision of a 24–32-nt oligonucleotide containing the lesion (
Within a DNA repair mechanism, the process of verifying a target site is of paramount importance, because this step affords a mechanism the high specificity that grants efficient processing of cytotoxic or carcinogenic DNA lesions while preventing futile repair. Crystal structures of XPD from different archaeal organisms have provided valuable insight into the general architecture of this enzyme (
). In our studies, we used XPD from the archaeal organism Thermoplasma acidophilum (taXPD). The enzyme consists of four domains: two RecA-like helicase domains, a domain coordinating an iron-sulfur cluster, and an arch domain. The iron-sulfur and arch domains together with helicase domain 1 comprise a narrow pore with ∼1-nm diameter (
). In addition, 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 (
). However, the exact mechanism of lesion verification by and in particular the impressive substrate versatility of XPD remained elusive so far.
We used the single molecule technique of atomic force microscopy (AFM) to directly visualize individual XPD-DNA complexes at nanometer resolution. By introducing a specific lesion at a known position in long DNA fragments (916 base pairs), we created substrates that more closely resemble the naturally occurring in vivo substrates than the short DNA oligonucleotides utilized with other methods for the analysis of protein-DNA interactions. Importantly, the exact knowledge of the lesion position within the DNA substrate allows us to distinguish between specifically 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. The lesions are representatives of two distinct classes of damages repaired by NER, a fluorescein as a representative for bulky DNA adducts (
). Our data clearly demonstrate specific stalling of taXPD at these target sites upon ATP-driven translocation on long DNA substrates. Most notably, however, 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.
Damage recognition and verification are critical processes in DNA repair, which have to ensure the speedy detection and processing of DNA lesions yet avoid futile incisions. The helicase XPD has been proposed to play a critical role in NER damage verification. Previous studies further indicated that XPD translocation may be stalled by DNA lesions, suggesting that the protein may be involved in damage recognition (
). Single molecule imaging by AFM allowed us to investigate the contributions of the XPD enzyme to the NER lesion recognition and verification process and to analyze this function in the presence of long DNA substrates (>900 bp), which mimic more closely the in vivo situation in the cell as compared with the short substrates required for other in vitro studies.
We analyzed the ability of taXPD to interact with various DNA substrates. ssDNA or ss/dsDNA junctions were bound with moderately high affinity (∼150 nm). Interestingly, the affinity to dsDNA (not containing any ssDNA regions) was only less than 1 order of magnitude weaker than to ssDNA (Table 2). Furthermore, our AFM data indicate that taXPD was not only able to bind to but also to translocate along dsDNA in the presence of ATP (Fig. 1). However, in the absence of a ssDNA region in the DNA substrate, lesions in the DNA were only poorly recognized by the enzyme (Fig. 2). It is therefore tempting to speculate that taXPD adopts at least two different binding modes upon DNA binding, in which only the second binding mode, which is induced by an initial interaction with ssDNA, is competent of dsDNA unwinding and supports successful lesion verification.
Most importantly, our analysis compared directly for the first time NER damage recognition and verification for different lesions. Introduction of a lesion into a DNA substrate within the context of an unpaired DNA region led to complex formation with high specificity both for a CPD and for a fluorescein lesion, which differ significantly in their structure. However, when the lesion was removed from the unpaired region and positioned 3′ or 5′ to this region, a clear distinction in damage recognition became apparent (model shown in Fig. 4). A bulky fluorescein adduct leads to a stalled taXPD-DNA complex when the lesion is located on the translocating strand. In contrast, a CPD lesion is preferentially recognized when it is located on the opposite strand, i.e., the nontranslocating strand.
Differences in NER mechanistic details may be related to the strong observed dependence of DNA repair efficiencies on the degree of DNA helix destabilization by different lesions (
). A possible explanation for the strand selectivity observed in our studies is therefore a different recognition mechanism based on the diverse structural prerequisites for different types of lesions. In our studies, the bulky fluorescein adduct may result in direct mechanical blocking of XPD translocation, but only when it is encountered on the actual strand that the protein “holds on to.” Importantly, under the conditions used in our experiments, the fluorescein adduct is most likely negatively charged and would therefore not destabilize the negatively charged DNA duplex via intercalation. Loading of XPD onto DNA carrying the type of lesion represented by a fluorescein hence requires the presence of a DNA bubble (Fig. 2), which is provided in vivo by the concerted action of XPC and XPB. A CPD lesion also does not lead to a major destabilization of the DNA double helix (
). However, distortion of the CPD containing ssDNA strand by the thymine dimer may be sufficient to provide an access site for XPD loading, resulting in slightly enhanced XPD localization to the lesion site in the absence of a DNA bubble (Fig. 2). When loaded at an access site (DNA bubble) at a distance from the lesion, translocation of taXPD along the lesion-containing strand appears to be feasible and is not strongly hindered by the presence of the lesion. Therefore a different, so far unidentified verification process could be envisioned. It is tempting to speculate that, when the lesion is located on the nontranslocated strand, it may be in close proximity to the iron-sulfur cluster (Fig. 4A), thus supporting the hypothesis that the iron-sulfur cluster may act as a damage detector, as has been shown for other protein systems containing such clusters (
), who reported stalling of the archaeal Ferroplasma acidarmanus XPD helicase by a CPD lesion located both in the nontranslocated and in the translocated DNA strand. Their studies provide support for the formation of a stable complex between F. acidarmanus XPD helicase and a CPD lesion in the translocated DNA strand, with interactions that are strong enough to withstand incision by the CPD processing glycosylase T4 Endo V. Both mechanistic deviations between XPD from different species and/or variations caused by different experimental approaches are conceivable and will be worth investigating in future studies. Notably, compared with the short oligonucleotides employed in these biochemical experiments, the long DNA substrates in our AFM studies provide better stability of the DNA duplex and, importantly, more closely resemble physiological conditions. Our data do not argue against such stable complex formation with CPD lesions in the translocated strand, but report a strong preference for detection of CPD lesions in the nontranslocated strand versus in the translocated strand. Importantly, once verified, lesion processing by taXPD appears to be similar for all substrates in our experiments, as suggested by vast differences in lesion specificities (Fig. 2) but comparable bend angle distributions for complexes engaged at specific lesion sites (Fig. 3).
In the context of DNA damage search, taXPD clearly requires a ssDNA region for successful stalling at a lesion site. In eukaryotic NER, XPD is part of the TFIIH complex, which is initially recruited to the damaged DNA by XPC. The presence of a destabilized DNA region (as for instance in a 3-nt DNA bubble) has been shown to be essential for XPC-induced loading of NER factors and the subsequent excision of a CPD lesion (
) show convincing evidence for recognition of CPD damages in the same DNA strand that the TFIIH complex is loaded on, these studies do not exclude incision competent recognition of CPD on the nontranslocated strand after XPC loading on a symmetrical DNA bubble 3′ to the lesion. Importantly, their data further corroborate a two-step (bipartite) model and the importance of TFIIH orientation for correct lesion recognition and processing.
Once taXPD has verified the presence of an NER target, processing of the lesion involves conformational changes in the stalled complex at the lesion visible by a significant shift in the maximum of the distribution of induced DNA bend angles at the site of the bound protein from ∼50° to ∼65° (Fig. 3). These transitions were independent of the lesion type and of the details of the preceding lesion recognition strategy. In the context of the eukaryotic repair cascade, this conformational change may be the prerequisite for the recruitment of the remaining NER machinery, including the endonucleases XPG and XPF for damage removal. This conformational shift is completely absent in samples of the taXPD variant K170A, which is incapable of detecting NER target sites. These results further underline the significance of stalled XPD translocation for concomitant lesion recognition as a prerequisite for lesion specific processing by XPD in NER. Fluorescein- and CPD-DNA structures have previously been shown to display intrinsic bending by 15–30° at the lesion (
). However, AFM bend angle distributions obtained on the lesion sites in the absence of protein (supplemental Fig. S6) contain no major population displaying these bend angles and hence argue against an innate preformed DNA conformation that XPD binds to. It is conceivable that the ∼30° bend angle conformation observed in all specific site bend angle distributions may represent a complex conformation sampled by the protein on the path to the specific lesion repair signaling complex. However, it is the larger bend angle state (∼65°) that is dominant in the lesion-specific complexes (∼70% of all complexes) and comparable in population to the significantly less bent state (∼50°) in the nonspecific complexes at homoduplex DNA sites. We therefore interpret this bend angle conformation as the specific, lesion associated state competent for induction of subsequent DNA repair events. Interestingly, the conformational changes occurred in the presence of either ATP or ATPγS, indicating that the lesion-dependent rearrangement of the taXPD-DNA complex involves ATP binding but not hydrolysis. The requirement of ATP rebinding for lesion-dependent conformational changes is strongly reminiscent of the prokaryotic NER mechanism. In the prokaryotic NER damage search and recognition complex, UvrB is thought to undergo initial conformational changes upon ATP hydrolysis. This process leads to its localization at the lesion site, followed by ATP rebinding and concomitant formation of a stable, specific preincision complex at the lesion (
), despite a complete lack of sequence and structural homology between the involved enzymes. This is the first example showing that an individual step within the verification process in the NER cascade may be strikingly similar between prokaryotic and eukaryotic NER, corroborating the conservation of this biologically essential DNA repair system.
We thank Samuel Wilson for providing the modified DNA plasmid for DNA substrate preparation, Stefanie Wolski for assistance with protein preparation, Jochen Kuper for preparation of the XPD-DNA model in Fig. 4A, and Hermann Schindelin and Jochen Kuper for critical reading of the manuscript.