NMR study on the interaction between RPA and DNA decamer containing cis-syn cyclobutane pyrimidine dimer in the presence of XPA: implication for damage verification and strand-specific dual incision in nucleotide excision repair

In mammalian cells, nucleotide excision repair (NER) is the major pathway for the removal of bulky DNA adducts. Many of the key NER proteins are members of the XP family (XPA, XPB, etc.), which was named on the basis of its association with the disorder xerodoma pigmentosum. Human replication protein A (RPA), the ubiquitous single-stranded DNA-binding protein, is another of the essential proteins for NER. RPA stimulates the interaction of XPA with damaged DNA by forming an RPA–XPA complex on damaged DNA sites. Binding of RPA to the undamaged DNA strand is most important during NER, because XPA, which directs the excision nucleases XPG and XPF, must bind to the damaged strand. In this study, nuclear magnetic resonance (NMR) spectroscopy was used to assess the binding of the tandem high affinity DNA-binding domains, RPA-AB, and of the isolated domain RPA-A, to normal DNA and damaged DNA containing the cyclobutane pyrimidine dimer (CPD) lesion. Both RPA-A and RPA-AB were found to bind non- specifically to both strands of normal and CPD- containing DNA duplexes. There were no differences observed when binding to normal DNA duplex was examined in the presence of the minimal DNA-binding domain of XPA (XPA-MBD). However, there is a drastic difference for CPD-damaged DNA duplex as both RPA-A and RPA-AB bind specifically to the undamaged strand. The strand-specific binding of RPA and XPA to the damaged duplex DNA shows that RPA and XPA play crucial roles in damage verification and guiding cleavage of damaged DNA during NER.     

Interactions of human nucleotide excision repair protein XPA with DNA and RPA70 Delta C327: chemical shift mapping and 15N NMR relaxation studies

Human XPA is an essential component in the multienzyme nucleotide excision repair (NER) pathway. The solution structure of the minimal DNA binding domain of XPA (XPA-MBD: M98-F219) was recently determined [Buchko et al. (1998) Nucleic Acids Res. 26, 2779-2788, Ikegami et al. (1998) Nat. Struct. Biol. 5, 701-706] and shown to consist of a compact zinc-binding core and a loop-rich C-terminal subdomain connected by a linker sequence. Here, the solution structure of XPA-MBD was further refined using an entirely new class of restraints based on pseudocontact shifts measured in cobalt-substituted XPA-MBD. Using this structure, the surface of XPA-MBD which interacts with DNA and a fragment of the largest subunit of replication protein A (RPA70 Delta C327: M1-Y326) was determined using chemical shift mapping. DNA binding in XPA-MBD was highly localized in the loop-rich subdomain for DNA with or without a lesion [dihydrothymidine (dhT) or 6-4-thymidine-cytidine (64TC)], or with DNA in single- or double-stranded form, indicating that the character of the lesion itself is not the driving force for XPA binding DNA. RPA70 Delta C327 was found to contact regions in both the zinc-binding and loop-rich subdomains. Some overlap of the DNA and RPA70 Delta C327 binding regions was observed in the loop-rich subdomain, indicating a possible cooperative DNA-binding mode between XPA and RPA70 Delta C327. To complement the chemical shift mapping data, the backbone dynamics of free XPA-MBD and XPA-MBD bound to DNA oligomers containing dhT or 64TC lesions were investigated using 15N NMR relaxation data. The dynamic analyses for the XPA-MBD complexes with DNA revealed localized increases and decreases in S2 and an increase in the global correlation time. Regions of XPA-MBD with the largest increases in S2 overlapped regions having the largest chemical shifts changes upon binding DNA, indicating that the loop-rich subdomain becomes more rigid upon binding DNA. Interestingly, S2 decreased for some residues in the zinc-binding core upon DNA association, indicating a possible concerted structural rearrangement on binding DNA.     

Preferential binding of human full-length XPA and the minimal DNA binding domain (XPA-MF122) with the mitomycin C-DNA interstrand cross-link

Nucleotide excision repair (NER) is an important cellular mechanism that removes radiation-induced and chemically induced damage from DNA. The XPA protein is involved in the damage recognition step of NER and appears to function by binding damaged DNA and recruiting other proteins to the site. It may also play a role in subsequent steps of NER through interaction with other repair proteins. Interstrand cross-links are of particular interest, since these lesions involve both strands of duplex DNA and present special challenges to the repair machinery. Using 14 and 25 bp duplex oligonucleotides containing a defined, well-characterized single mitomycin C (MMC)-DNA interstrand cross-link, we have shown through gel shift analysis that both XPA and a minimal DNA binding domain of XPA (XPA-MF122) preferentially bind to MMC-cross-linked DNA with a greater specificity and a higher affinity (>2-fold) than to the same undamaged DNA sequence. This preferential binding to MMC-cross-linked DNA occurs in the absence of other proteins from the NER complex. Differences in binding affinity and specificity were observed among the different protein-DNA combinations that were both protein and DNA specific. Defining XPA-MMC-DNA interactions may aid in elucidating the mechanism by which DNA cross-links and other forms of DNA damage are recognized and repaired by the NER machinery in eukaryotic cells.     

Crosslinking of nucleotide excision repair proteins with DNA containing photoreactive damages

Photoreactive DNA duplexes mimicking substrates of nucleotide excision repair (NER) system were used to analyze the interaction of XPC-HR23B, RPA, and XPA with damaged DNA. Photoreactive groups in one strand of DNA duplex (arylazido-dCMP or 4-thio-dUMP) were combined with anthracenyl-dCMP residue at the opposite strand to analyze contacts of NER factors with damaged and undamaged strands. Crosslinking of XPC-HR23B complex with photoreactive 48-mers results in modification of XPC subunit. XPC-HR23B did not crosslink with DNA duplex bearing bulky residues in both strands while this modification does not prevent interaction of DNA with XPA. The data on crosslinking of XPA and RPA with photoreactive DNA duplexes containing bulky group in one of the strands are in favor of XPA preference to interact with the damaged strand and RPA preference for the undamaged strand. The results support the understanding and set the stage for dynamically oriented experiments of how the pre-incision complex is formed in the early stage of NER.     

Strand-specific binding of RPA and XPA to damaged duplex DNA

The nucleotide excision repair (NER) pathway is a major pathway used to repair bulky adduct DNA damage. Two proteins, xeroderma pigmentosum group A protein (XPA) and replication protein A (RPA), have been implicated in the role of DNA damage recognition in the NER pathway. The particular manner in which these two damage recognition proteins align themselves with respect to a damaged DNA site was assessed using photoreactive base analogues within specific DNA substrates to allow site-specific cross-linking of the damage recognition proteins. Results of these studies demonstrate that both RPA and XPA are in close proximity to the adduct as measured by cross-linking of each protein directly to the platinum moiety. Additional studies demonstrate that XPA contacts both the damaged and undamaged strands of the duplex DNA. Direct evidence is presented demonstrating preferential binding of RPA to the undamaged strand of a duplex damaged DNA molecule.     

Localization of xeroderma pigmentosum group A protein and replication protein A on damaged DNA in nucleotide excision repair

The interaction of xeroderma pigmentosum group A protein (XPA) and replication protein A (RPA) with damaged DNA in nucleotide excision repair (NER) was studied using model dsDNA and bubble-DNA structure with 5-{3-[6-(carboxyamido-fluoresceinyl)amidocapromoyl]allyl}-dUMP lesions in one strand and containing photoreactive 5-iodo-dUMP residues in defined positions. Interactions of XPA and RPA with damaged and undamaged DNA strands were investigated by DNA–protein photocrosslinking and gel shift analysis. XPA showed two maximums of crosslinking intensities located on the 5′-side from a lesion. RPA mainly localized on undamaged strand of damaged DNA duplex and damaged bubble-DNA structure. These results presented for the first time the direct evidence for the localization of XPA in the 5′-side of the lesion and suggested the key role of XPA orientation in conjunction with RPA binding to undamaged strand for the positioning of the NER preincision complex. The findings supported the mechanism of loading of the heterodimer consisting of excision repair cross-complementing group 1 and xeroderma pigmentosum group F proteins by XPA on the 5′-side from the lesion before damaged strand incision. Importantly, the proper orientation of XPA and RPA in the stage of preincision was achieved in the absence of TFIIH and XPG.     

Replication protein A interactions with DNA. III. Molecular basis of recognition of damaged DNA

Human replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein (subunits of 70, 32, and 14 kDa) that is required for cellular DNA metabolism. RPA has been reported to interact specifically with damaged double-stranded DNA and to participate in multiple steps of nucleotide excision repair (NER) including the damage recognition step. We have examined the mechanism of RPA binding to both single-stranded and double-stranded DNA (ssDNA and dsDNA, respectively) containing damage. We show that the affinity of RPA for damaged dsDNA correlated with disruption of the double helix by the damaged bases and required RPAs ssDNA-binding activity. We conclude that RPA is recognizing single-stranded character caused by the damaged nucleotides. We also show that RPA binds specifically to damaged ssDNA. The specificity of binding varies with the type of damage with RPA having up to a 60-fold preference for a pyrimidine(6-4)pyrimidone photoproduct. We show that this specific binding was absolutely dependent on the zinc-finger domain in the C-terminus of the 70-kDa subunit. The affinity of RPA for damaged ssDNA was 5 orders of magnitude higher than that of the damage recognition protein XPA (xeroderma pigmentosum group A protein). These findings suggest that RPA probably binds to both damaged and undamaged strands in the NER excision complex. RPA binding may be important for efficient excision of damaged DNA in NER.     

Structural basis for the recruitment of ERCC1-XPF to nucleotide excision repair complexes by XPA

The nucleotide excision repair (NER) pathway corrects DNA damage caused by sunlight, environmental mutagens and certain antitumor agents. This multistep DNA repair reaction operates by the sequential assembly of protein factors at sites of DNA damage. The efficient recognition of DNA damage and its repair are orchestrated by specific protein-protein and protein-DNA interactions within NER complexes. We have investigated an essential protein-protein interaction of the NER pathway, the binding of the XPA protein to the ERCC1 subunit of the repair endonuclease ERCC1-XPF. The structure of ERCC1 in complex with an XPA peptide shows that only a small region of XPA interacts with ERCC1 to form a stable complex exhibiting submicromolar binding affinity. However, this XPA peptide is a potent inhibitor of NER activity in a cell-free assay, blocking the excision of a cisplatin adduct from DNA. The structure of the peptide inhibitor bound to its target site reveals a binding interface that is amenable to the development of small molecule peptidomimetics that could be used to modulate NER repair activities in vivo.     

RPA repair recognition of DNA containing pyrimidines bearing bulky adducts

Recognition of new DNA nucleotide excision repair (NER) substrate analogs, 48-mer ddsDNA (damaged double-stranded DNA), by human replication protein A (hRPA) has been analyzed using fluorescence spectroscopy and photoaffinity modification. The aim of the present work was to find quantitative characteristics of RPA-ddsDNA interaction and RPA subunits role in this process. The designed DNA structures bear bulky substituted pyrimidine nitrogen bases at the inner positions of duplex forming DNA chains. The photoreactive 4-azido-2,5-difluoro-3- pyridin-6-yl (FAP) and fluorescent antracenyl, pyrenyl (Antr, Pyr) groups were introduced via different linker fragments into exo-4N of deoxycytidine or 5C of deoxyuridine. J-dU-containing DNA was used as a photoactive model of undamaged DNA strands. The reporter group was a fluorescein residue, introduced into the 5'-phosphate end of one duplex-forming DNA strand. RPA-dsDNA association constants and the molar RPA/dsDNA ratio have been calculated based on fluorescence anisotropy measurements under conditions of a 1:1 RPA/dsDNA molar ratio in complexes. The evident preference for RPA binding to ddsDNA over undamaged dsDNA distinctly depends on the adduct type and varies in the following way: undamaged dsDNA < Antr-dC-ddsDNA < mmdsDNA < FAPdU-, Pyr-dU-ddsDNA < FAP-dC-ddsDNA (K(D) = 68 +/- 1; 25 +/- 6; 13 +/- 1; 8 +/- 2, and 3.5 +/- 0.5 nM correspondingly) but weakly depends on the chain integrity. Interestingly the bulkier lesions not in all cases have a greater effect on RPA affinity to ddsDNA. The experiments on photoaffinity modification demonstrated only p70 of compactly arranged RPA directly interacting with dsDNA. The formation of RPA-ddsDNA covalent adducts was drastically reduced when both strands of DNA duplex contained virtually opposite located FAP-dC and Antr-dC. Thus RPA requires undamaged DNA strand presence for the effective interaction with dsDNA bearing bulky damages and demonstrates the early NER factors characteristic features underlying strand discrimination capacity and poor activity of the NER system toward double damaged DNA.     

Human nucleotide excision repair protein XPA: NMR spectroscopic studies of an XPA fragment containing the ERCC1-binding region and the minimal DNA-binding domain (M59-F219)

XPA is a central protein component of nucleotide excision repair (NER), a ubiquitous, multi-component cellular pathway responsible for the removal and repair of many structurally distinct DNA lesions from the eukaryotic genome. The solution structure of the minimal DNA-binding domain of XPA (XPA-MBD: M98-F219) has recently been determined and chemical shift mapping experiments with 15N-labeled XPA-MBD show that XPA binds DNA along a basic surface located in the C-terminal loop-rich subdomain. Here, XPA-DNA interactions are further characterized using an XPA fragment containing the minimal DNA-binding domain plus the ERCC1-binding region (XPA-EM: M59-F219). The 15N/1H HSQC spectrum of XPA-EM closely maps onto the 15N/1H HSQC spectrum of XPA-MBD, suggesting the DNA-binding domain is intact in the larger XPA fragment. Such a conclusion is corroborated by chemical shift mapping experiments of XPA-EM with a single strand DNA oligomer, dCCAATAACC (d9), that show the same set of 15N/1H HSQC cross peaks are effected by the addition of DNA. However, relative to DNA-free XPA-MBD, the 15N/1H HSQC cross peaks of many of the basic residues in the loop-rich subdomain of DNA-free XPA-EM are less intense, or gone altogether, suggesting the acidic ERRC1-binding region of XPA-EM may associate transiently with the basic DNA-binding surface. While the DNA-binding domain in XPA-EM is structured and functional, 15N-edited NOESY spectra of XPA-EM indicate that the acidic ERRC1-binding region is unstructured. If the structural features observed for XPA-EM persist in XPA, transient intramolecular association of the ERCC1-binding domain with the DNA-binding region may play a role in the sequential assembly of the NER components.     

Equilibrium and stop-flow kinetic studies of fluorescently labeled DNA substrates with DNA repair proteins XPA and replication protein A.

Nucleotide excision repair (NER) is a crucial pathway in the maintenance of genome stability requiring at least two dozen proteins. XPA and RPA have essential roles in the damage recognition step of NER. To better understand the mechanism of their interactions with DNA, we utilized equilibrium and stop-flow kinetic approaches with fluorescently labeled oligonucleotides. Fluorescein is a bona fide NER lesion because a circular plasmid with a single defined fluorescein was repaired by efficient extracts from Xenopus oocyte nuclei. Single-stranded and double-stranded oligonucleotides 5'-labeled with fluorescein were used in the subsequent studies. Oligonucleotide fluorescence was quenched upon specific binding to full-length recombinant Xenopus XPA (xXPA) and/or human RPA. The binding was highly sensitive to the buffer conditions. Analysis of equilibrium binding data with ds DNA and xXPA revealed a single dissociation constant (K(d)) of 24.4 nM. Stopped-flow kinetic experiments were described by a first-order on-rate constant k(on) of 9.03 x 10(8) M(-1) s(-1) and k(off) of 26.1 s(-1). From the ratio of off-rate to on-rate, a calculated K(d) of 28.9 nM was obtained, revealing that the kinetic and equilibrium studies were consistent. The affinity of xXPA for ds undamaged DNA determined in our spectrofluorometry experiments was up to 3 orders of magnitude higher than previously reported values using different substrates, conditions, and assays [gel-shifts (EMSA), filter-binding, anisotropy, and surface plasmon resonance]. The same substrate DNA containing a 4-bp mismatch in the middle yielded a K(d) five times higher (158 nM), indicating weaker binding by xXPA. The differences in K(d) values for these two substrates were mainly attributable to the k(on), rather than k(off) rates. Fluorescence intensity changes upon interaction of xXPA with ss 50-mer were too low to calculate an accurate K(d). Although recombinant human RPA binding to the ds 50-mer was very weak (K(d) > 1 mM), stop-flow and equilibrium measurements to ss oligonucleotide yielded K(d) values of 96 and 20.3 nM, respectively, which correlated with previously reported values using gel mobility shift assays and a similarly sized poly-dT. Equilibrium and stop-flow measurements to the cognate and mismatched ds oligonucleotides using both xXPA and hRPA yielded a 2- to 3-fold increase in the K(d).     

Human nucleotide excision repair protein XPA: extended X-ray absorption fine-structure evidence for a metal-binding domain.

The ubiquitous, multi-enzyme, nucleotide excision repair (NER) pathway is responsible for correcting a wide range of chemically and structurally distinct DNA lesions in the eukaryotic genome. Human XPA, a 31 kDa, zinc-associated protein, is thought to play a major NER role in the recognition of damaged DNA and the recruitment of other proteins, including RPA, ERCC1, and TFIIH, to repair the damage. Sequence analyses and genetic evidence suggest that zinc is associated with a C4-type motif, C105-X2-C108-X17-C126-X2-C129, located in the minimal DNA binding region of XPA (M98-F219). The zinc-associated motif is essential for damaged DNA recognition. Extended X-ray absorption fine structure (EXAFS) spectra collected on the zinc associated minimal DNA-binding domain of XPA (ZnXPA-MBD) show directly, for the first time, that the zinc is coordinated to the sulfur atoms of four cysteine residues with an average Zn-S bond length of 2.34+/-0.01 A. XPA-MBD was also expressed in minimal medium supplemented with cobalt nitrate to yield a blue-colored protein that was primarily (>95%) cobalt associated (CoXPA-MBD). EXAFS spectra collected on CoXPA-MBD show that the cobalt is also coordinated to the sulfur atoms of four cysteine residues with an average Co-S bond length of 2.33+/-0.02 A.     

Preferential binding of human XPA to the mitomycin C-DNA interstrand crosslink and modulation by arsenic and cadmium

The Xeroderma Pigmentosum A (XPA) protein is involved in the DNA damage recognition and repair complex formation steps of nucleotide excision repair (NER), and has been shown to preferentially bind to various forms of DNA damage including bulky lesions. DNA interstrand crosslinks are of particular interest as a form of DNA damage, since these lesions involve both strands of duplex DNA and present special challenges to the repair machinery, and mitomycin C (MMC) is one of several useful cancer chemotherapy drugs that induce these lesions. Purified XPA and the minimal DNA-binding domain of XPA are both fully capable of preferentially binding to MMC-DNA interstrand crosslinks in the absence of other proteins from the NER complex. Circular dichroism (CD) and gel shift assays were used to investigate XPA-DNA binding and to assess changes in secondary structure induced as a consequence of the interaction of XPA with model MMC-crosslinked and unmodified DNAs. These studies revealed that while XPA demonstrates only a modest increase in affinity for adducted DNA, it adopts a different conformation when bound to MMC-damaged DNA than when bound to undamaged DNA. This change in conformation may be more important in recruiting other proteins into a competent NER complex at damaged sites than preferential binding per se. Arsenic had little effect on XPA binding even at toxic concentrations, whereas cadmium reduced XPA binding to DNA to 10-15% that of Zn-XPA, and zinc addition could only partially restore activity. In addition, there was little or no change in conformation when Cd-XPA bound MMC-crosslinked DNA even though it demonstrated preferential binding, which may contribute to the mechanism by which cadmium can act as a co-mutagen and co-carcinogen.     

Interactions of mammalian proteins with cisplatin-damaged DNA

We have undertaken the systematic isolation and characterization of mammalian proteins which display an affinity for cisplatin-damaged DNA. Fractionation of human cell extracts has led to the identification of two classes of proteins. The first includes proteins that bind duplex DNA in the absence of cisplatin damage and retain their affinity for DNA in the presence of cisplatin-DNA adducts. The DNA-dependent protein kinase (DNA-PK) falls into this class. The inhibition of DNA-PK phosphorylation activity by cisplatin-damaged DNA has led to the hypothesis that cisplatin sensitization of mammalian cells to ionizing radiation may be mediated by DNA-PK. The second class of proteins identified are those which display a high relative affinity for cisplatin-damaged DNA and a low affinity for undamaged duplex DNA. Proteins that fall into this class include high mobility group 1 protein (HMG-1), replication protein A (RPA) and xeroderma pigmentosum group A protein (XPA). Each protein has been isolated and purified in the lab. The interaction of each protein with cisplatin-damaged DNA has been assessed in electrophoretic mobility shift assays. A series of DNA binding experiments suggests that RPA binds duplex DNA via denaturation and subsequent preferential binding to the undamaged DNA strand of the partial duplex. DNA substrates prepared with photo-reactive base analogs on either the damaged or undamaged DNA strand have also been employed to investigate the mechanism and specific protein-DNA interactions that occur as each protein binds to cisplatin-damaged DNA. Results suggest both damage and strand specificity for RPA and XPA binding cisplatin-damaged DNA.     

Nucleotide excision repair by mutant xeroderma pigmentosum group A (XPA) proteins with deficiency in interaction with RPA

The xeroderma pigmentosum group A protein (XPA) is a core component of nucleotide excision repair (NER). To coordinate early stage NER, XPA interacts with various proteins, including replication protein A (RPA), ERCC1, DDB2, and TFIIH, in addition to UV-damaged or chemical carcinogen-damaged DNA. In this study, we investigated the effects of mutations in the RPA binding regions of XPA on XPA function in NER. XPA binds through an N-terminal region to the middle subunit (RPA32) of the RPA heterotrimer and through a central region that overlaps with its damaged DNA binding region to the RPA70 subunit. In cell-free NER assays, an N-terminal deletion mutant of XPA showed loss of binding to RPA32 and reduced DNA repair activity, but it could still bind to UV-damaged DNA and RPA. In contrast, amino acid substitutions in the central region reduced incisions at the damaged site in the cell-free NER assay, and four of these mutants (K141A, T142A, K167A, and K179A) showed reduced binding to RPA70 but normal binding to damaged DNA. Furthermore, mutants that had one of the four aforementioned substitutions and an N-terminal deletion exhibited lower DNA incision activity and binding to RPA than XPA with only one of these substitutions or the deletion. Taken together, these results indicate that XPA interaction with both RPA32 and RPA70 is indispensable for NER reactions.     

Binding of XPA and RPA to damaged DNA investigated by fluorescence anisotropy

The proteins XPA and RPA are assumed to be involved in primary damage recognition of global genome nucleotide excision repair. XPA as well as RPA have been each reported to specifically bind DNA lesions, and ternary complex formation with damaged DNA has also been shown. We employed fluorescence anisotropy measurements to study the DNA-binding properties of XPA and RPA under true equilibrium conditions using damaged DNA probes carrying a terminal fluorescein modification as a reporter. XPA binds with low affinity and in a strongly salt-dependent manner to DNA containing a 1,3-d(GTG) intrastrand adduct of the anticancer drug cisplatin or a 6-nt mismatch (K(D) = 400 nM) with 3-fold preference for damaged vs undamaged DNA. At near physiological salt conditions binding is very weak (K(D) > 2 microM). RPA binds to damaged DNA probes with dissociation constants in the range of 20 nM and a nearly 15-fold preference over undamaged DNA. The presence of a cisplatin modification weakens the affinity of RPA for single-stranded DNA by more than 1 order of magnitude indicating that binding to the lesion itself is not a driving force in damage recognition. Our fluorescence anisotropy assays also show that the presence of XPA does not enhance the affinity of RPA for damaged DNA although both proteins interact. In contrast, cooperative binding of XPA and RPA is observed in EMSA. Our results point to a damage-sensing function of the XPA-RPA complex with RPA mediating the important DNA contacts.     

Multiple DNA binding domains mediate the function of the ERCC1-XPF protein in nucleotide excision repair

ERCC1-XPF is a heterodimeric, structure-specific endonuclease that cleaves single-stranded/double-stranded DNA junctions and has roles in nucleotide excision repair (NER), interstrand crosslink (ICL) repair, homologous recombination, and possibly other pathways. In NER, ERCC1-XPF is recruited to DNA lesions by interaction with XPA and incises the DNA 5' to the lesion. We studied the role of the four C-terminal DNA binding domains in mediating NER activity and cleavage of model substrates. We found that mutations in the helix-hairpin-helix domain of ERCC1 and the nuclease domain of XPF abolished cleavage activity on model substrates. Interestingly, mutations in multiple DNA binding domains were needed to significantly diminish NER activity in vitro and in vivo, suggesting that interactions with proteins in the NER incision complex can compensate for some defects in DNA binding. Mutations in DNA binding domains of ERCC1-XPF render cells more sensitive to the crosslinking agent mitomycin C than to ultraviolet radiation, suggesting that the ICL repair function of ERCC1-XPF requires tighter substrate binding than NER. Our studies show that multiple domains of ERCC1-XPF contribute to substrate binding, and are consistent with models of NER suggesting that multiple weak protein-DNA and protein-protein interactions drive progression through the pathway. Our findings are discussed in the context of structural studies of individual domains of ERCC1-XPF and of its role in multiple DNA repair pathways.     

A new structural insight into XPA–DNA interactions

XPA (xeroderma pigmentosum group A) protein is an essential factor for NER (nucleotide excision repair) which is believed to be involved in DNA damage recognition/verification, NER factor recruiting and stabilization of repair intermediates. Past studies on the structure of XPA have focused primarily on XPA interaction with damaged DNA. However, how XPA interacts with other DNA structures remains unknown though recent evidence suggest that these structures could be important for its roles in both NER and non-NER activities. Previously, we reported that XPA recognizes undamaged DNA ds/ssDNA (double-strand/single-strandDNA) junctions with a binding affinity much higher than its ability to bind bulky DNA damage. To understand how this interaction occurs biochemically we implemented a structural determination of the interaction using a MS-based protein footprinting method and limited proteolysis. By monitoring surface accessibility of XPA lysines to NHS-biotin modification in the free protein and the DNA junction-bound complex we show that XPA physically interacts with the DNA junctions via two lysines, K168 and K179, located in the previously known XPA(98–219) DBD (DNA-binding domain). Importantly, we also uncovered new lysine residues, outside of the known DBD, involved in the binding. We found that residues K221, K222, K224 and K236 in the C-terminal domain are involved in DNA binding. Limited proteolysis analysis of XPA–DNA interactions further confirmed this observation. Structural modelling with these data suggests a clamp-like DBD for the XPA binding to ds/ssDNA junctions. Our results provide a novel structure-function view of XPA–DNA junction interactions.     

The limited strand-separating activity of the UvrAB protein complex and its role in the recognition of DNA damage.

The recognition by Escherichia coli Uvr nucleotide excision repair proteins of a variety of lesions with diverse chemical structures and the presence of helicase activity in the UvrAB complex which can displace short oligonucleotides annealed to single-stranded DNA led to a model in which this activity moves UvrAB along undamaged DNA to damaged sites where the lesion blocks further translocation and the protein-DNA pre-incision complex is formed. To evaluate this mechanism for damage recognition, we constructed substrates with oligonucleotides of different lengths annealed to single-stranded DNA circles and placed a single 2-(acetylamino)fluorene (AAF) lesion either on the oligonucleotide or on the circle. For the substrates with no lesion, the UvrAB complex effectively displaced a 22-mer but not a 27-mer or longer fragments. The presence of AAF on the oligonucleotide significantly increased the release of the 27-mer but oligomers of 30 or longer were not separated. Placing the lesion on the circular strand did not block the release of the fragments. Instead, the releasing activity of UvrAB was stimulated and also depended on the length of the annealed oligonucleotide. These observations do not agree with the predictions of a damage recognition mechanism that depends on helicase-driven translocation. Most likely, the strand-separating activity of UvrAB is a consequence of local changes occurring during the formation of a DNA-protein pre-incision complex at the damaged site and is not due to translocation of the protein along undamaged DNA to locate a lesion.