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.     

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.     

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.     

Effect of point substitutions within the minimal DNA-binding domain of xeroderma pigmentosum group a protein on interaction with DNA intermediates of nucleotide excision repair

Xeroderma pigmentosum factor A (XPA) is one of the key proteins in the nucleotide excision repair (NER) process. The effects of point substitutions in the DNA-binding domain of XPA (positively charged lysine residues replaced by negatively charged glutamate residues: XPA K204E, K179E, K141E, and tandem mutant K141E/K179E) on the inter-action of the protein with DNA structures modeling intermediates of the damage recognition and pre-incision stages in NER were analyzed. All these mutations decreased the affinity of the protein to DNA, the effect depending on the substitution and the DNA structure. The mutant as well as wild-type proteins bind with highest efficiency partly open damaged DNA duplex, and the affinity of the mutants to this DNA is reduced in the order: K204E > K179E ? K141E = K141/179E. For all the mutants, decrease in DNA binding efficiency was more pronounced in the case of full duplex and single-stranded DNA than with bubble-DNA structure, the difference between protein affinities to different DNA structures increasing as DNA binding activity of the mutant decreased. No effect of the studied XPA mutations on the location of the protein on the partially open DNA duplex was observed using photoinduced crosslinking with 5-I-dUMP in different positions of the damaged DNA strand. These results combined with earlier published data suggest no direct correlation between DNA binding and activity in NER for these XPA mutants.     

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.     

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.     

Cooperative interaction of human XPA stabilizes and enhances specific binding of XPA to DNA damage

Human xeroderma pigmentosum group A (XPA) is an essential protein for nucleotide excision repair (NER). We have previously reported that XPA forms a homodimer in the absence of DNA. However, what oligomeric forms of XPA are involved in DNA damage recognition and how the interaction occurs in terms of biochemical understanding remain unclear. Using the homogeneous XPA protein purified from baculovirus-infected Sf21 insect cells and the methods of gel mobility shift assays, gel filtration chromatography, and UV-cross-linking, we demonstrated that both monomeric and dimeric XPA bound to the DNA adduct of N-acetyl-2-aminofluorene (AAF), while showing little affinity for nondamaged DNA. The binding occurred in a sequential and protein concentration-dependent manner. At relatively low-protein concentrations, XPA formed a complex with DNA adduct as a monomer, while at the higher concentrations, an XPA dimer was involved in the specific binding. Results from fluorescence spectroscopic and competitive binding analyses indicated that the specific binding of XPA to the adduct was significantly facilitated and stabilized by the presence of the second XPA in a positive cooperative manner. This cooperative binding exhibited a Hill coefficient of 1.9 and the step binding constants of K(1) = 1.4 x 10(6) M(-)(1) and K(2) = 1.8 x 10(7) M(-)(1). When interaction of XPA and RPA with DNA was studied, even though binding of RPA-XPA complex to adducted DNA was observed, the presence of RPA had little effect on the overall binding efficiency. Our results suggest that the dominant form for XPA to efficiently bind to DNA damage is the XPA dimer. We hypothesized that the concentration-dependent formation of different types of XPA-damaged DNA complex may play a role in cellular regulation of XPA activity.     

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.     

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.     

RPA stabilizes the XPA-damaged DNA complex through protein-protein interaction

The xeroderma pigmentosum group A complementing protein (XPA) and eukaryotic replication protein A (RPA) are among the major damage-recognition proteins involved in the early stage of nucleotide excision repair (NER). XPA and RPA are able to bind damaged DNA independently, although RPA interaction stimulates XPA binding to damaged DNA [Li, L., Lu, X., Peterson, C. A., and Legerski, R. J. (1995) Mol. Cell. Biol. 15, 5396-5402 (1); Stigger, E., Drissi, R., and Lee, S.-H. (1998) J. Biol. Chem. 273, 9337-9343 (2)]. In this study, we used surface plasmon resonance (SPR) analysis to investigate the interaction of XPA and RPA with two major types of UV-damaged DNA: the (6-4) photoproduct and the cis-syn cyclobutane dimer of thymidine. Both XPA and RPA preferentially bind to (6-4) photoproduct-containing duplex DNA over cis-syn cyclobutane dimer-containing DNA. The binding of XPA to (6-4) photoproduct was weak (K(D) = 2.13 x 10(-)(8) M), whereas RPA showed a very stable interaction with (6-4) photoproduct (K(D) = 2. 02 x 10(-)(10) M). When XPA and RPA were incubated together, the stability of the XPA-damaged DNA interaction was significantly enhanced by wild-type RPA. On the other hand, mutant RPA (RPA:p34Delta33C) defective in its interaction with XPA failed to stabilize XPA-damaged DNA complex. Taken together, our results suggest that a role for RPA in UV-damage recognition is to stabilize XPA-damaged DNA complex through protein-protein interaction.     

A mutation in helicase motif III of E. coli RecG protein abolishes branch migration of Holliday junctions.

The RecG protein of Escherichia coli catalyses branch migration of Holliday junctions made by RecA and dissociates synthetic X junctions into duplex products in reactions that require hydrolysis of ATP. To investigate the mode of action of this enzyme a chromosomal mutation that inactivates recG (recG162) was cloned and sequenced. The recG162 mutation is a G:C to A:T transition, which produces an Ala428 to Val substitution in the protein. This change affects a motif (motif III) in the protein that is highly conserved in DNA and RNA helicases. RecG162 protein was purified and shown to retain the ability to bind synthetic X and Y junctions. However, it does not dissociate these junctions and fails to catalyse branch migration of Holliday junction intermediates purified from a RecA strand exchange reaction. RecG162 retains a DNA-dependent ATPase activity, but this is much reduced relative to the wild-type protein, especially with single-stranded DNA as a co-factor. These results suggest that branch migration by RecG is related to a junction-targeted DNA helicase activity.     

The MER3 DNA helicase catalyzes the unwinding of holliday junctions

The MER3 protein of Saccharomyces cerevisiae is required for crossover in meiosis and has been suggested to act at the initiation of homologous pairing and the resolution of Holliday junctions. The purified MER3 protein is a DNA helicase that translocates along single-stranded DNA in the 3' to 5' direction displacing annealed DNA fragments. Here, MER3 was found to be able to unwind various double-stranded DNA (dsDNA) substrates, including a 30-bp dsDNA with a 20-nucleotide 3'-overhang, a 30-bp dsDNA with a 20-nucleotide 5'-overhang, a 50-bp dsDNA with blunt ends, and a Holliday junction with 25-bp arms, each of which had a blunt end. Efficient unwinding of the 3'-overhang substrate appeared to initiate by the binding of MER3 to the 3' single-stranded tail in a reaction that required six or more unpaired bases. Unwinding of the blunt end and 5'-overhang substrates appeared to initiate at the blunt ends of these substrates. Unwinding of the Holliday junction was more efficient than the unwinding of the blunt and 5'-overhang substrates and was influenced by Mg(2+) concentrations that cause changes in the structure of the junction. Possible roles for Holliday junction unwinding in meiotic crossover are discussed.     

DNA damage recognition during nucleotide excision repair in mammalian cells

For the bulk of mammalian DNA, the core protein factors needed for damage recognition and incision during nucleotide excision repair (NER) are the XPA protein, the heterotrimeric RPA protein, the 6 to 9-subunit TFIIH, the XPC-hHR23B complex, the XPG nuclease, and the ERCC1-XPF nuclease. With varying efficiencies, NER can repair a very wide range of DNA adducts, from bulky helical distortions to subtle modifications on sugar residues. Several of the NER factors have an affinity for damaged DNA. The strongest binding factor appears to be XPC-hHR23B but preferential binding to damage is also a property of XPA, RPA, and components of TFIIH. It appears that in order to be repaired by NER, an adduct in DNA must have two features: it must create a helical distortion, and there must be a change in DNA chemistry. Initial recognition of the distortion is the most likely function for XPC-hHR23B and perhaps XPA and RPA, whereas TFIIH is well-suited to locate the damaged DNA strand by locating altered DNA chemistry that blocks translocation of the XPB and XPD components.     

Domains in the XPA protein important in its role as a processivity factor

XPA is a protein essential for nucleotide excision repair (NER) where it is thought to function in damage recognition/verification. We have proposed an additional role, that of a processivity factor, conferring a processive mechanism of action on XPF and XPG, the endonucleases, involved in NER. The present study was undertaken to examine the domain(s) in the XPA gene that are important for the ability of the XPA protein to function as a processivity factor. Using site-directed mutagenesis, mutations were created in several of the exons of XPA and mutant XPA proteins produced. The results showed that the DNA binding domain of XPA is critical for its ability to act as a processivity factor. Mutations in both the zinc finger motif and the large basic cleft in this domain eliminated the ability of XPA to confer a processive mechanism of action on the endonucleases involved in NER.     

Relative stabilities of DNA three-way, four-way and five-way junctions (multi-helix junction loops): unpaired nucleotides can be stabilizing or destabilizing.

Competition binding and UV melting studies of a DNA model system consisting of three, four or five mutually complementary oligonucleotides demonstrate that unpaired bases at the branch point stabilize three- and five-way junction loops but destabilize four-way junctions. The inclusion of unpaired nucleotides permits the assembly of five-way DNA junction complexes (5WJ) having as few as seven basepairs per arm from five mutually complementary oligonucleotides. Previous work showed that 5WJ, having eight basepairs per arm but lacking unpaired bases, could not be assembled [Wang, Y.L., Mueller, J.E., Kemper, B. and Seeman, N.C. (1991) Biochemistry, 30, 5667-5674]. Competition binding experiments demonstrate that four-way junctions (4WJ) are more stable than three-way junctions (3WJ), when no unpaired bases are included at the branch point, but less stable when unpaired bases are present at the junction. 5WJ complexes are in all cases less stable than 4WJ or 3WJ complexes. UV melting curves confirm the relative stabilities of these junctions. These results provide qualitative guidelines for improving the way in which multi-helix junction loops are handled in secondary structure prediction programs, especially for single-stranded nucleic acids having primary sequences that can form alternative structures comprising different types of junctions.     

Xeroderma pigmentosum group A protein loads as a separate factor onto DNA lesions

Nucleotide excision repair (NER) is the main DNA repair pathway in mammals for removal of UV-induced lesions. NER involves the concerted action of more than 25 polypeptides in a coordinated fashion. The xeroderma pigmentosum group A protein (XPA) has been suggested to function as a central organizer and damage verifier in NER. How XPA reaches DNA lesions and how the protein is distributed in time and space in living cells are unknown. Here we studied XPA in vivo by using a cell line stably expressing physiological levels of functional XPA fused to green fluorescent protein and by applying quantitative fluorescence microscopy. The majority of XPA moves rapidly through the nucleoplasm with a diffusion rate different from those of other NER factors tested, arguing against a preassembled XPA-containing NER complex. DNA damage induced a transient ( approximately 5-min) immobilization of maximally 30% of XPA. Immobilization depends on XPC, indicating that XPA is not the initial lesion recognition protein in vivo. Moreover, loading of replication protein A on NER lesions was not dependent on XPA. Thus, XPA participates in NER by incorporation of free diffusing molecules in XPC-dependent NER-DNA complexes. This study supports a model for a rapid consecutive assembly of free NER factors, and a relatively slow simultaneous disassembly, after repair.     

The phage T4 protein UvsW drives Holliday junction branch migration

The phage T4 UvsW protein has been shown to play a crucial role in the switch from origin-dependent to recombination-dependent replication in T4 infections through the unwinding of origin R-loop initiation intermediates. UvsW also functions with UvsX and UvsY to repair damaged DNA through homologous recombination, and, based on genetic evidence, has been proposed to act as a Holliday junction branch migration enzyme. Here we report the purification and characterization of UvsW. Using oligonucleotide-based substrates, we confirm that UvsW unwinds branched DNA substrates, including X and Y structures, but shows little activity in unwinding linear duplex substrates with blunt or single-strand ends. Using a novel Holliday junction-containing substrate, we also demonstrate that UvsW promotes the branch migration of Holliday junctions efficiently through more than 1000 bp of DNA. The ATP hydrolysis-deficient mutant protein, UvsW-K141R, is unable to promote Holliday junction branch migration. However, both UvsW and UvsW-K141R are capable of stabilizing Holliday junctions against spontaneous branch migration when ATP is not present. Using two-dimensional agarose gel electrophoresis we also show that UvsW acts on T4-generated replication intermediates, including Holliday junction-containing X-shaped intermediates and replication fork-shaped intermediates. Taken together, these results strongly support a role for UvsW in the branch migration of Holliday junctions that form during T4 recombination, replication, and repair.     

Hexapeptides That Inhibit Processing of Branched DNA Structures Induce a Dynamic Ensemble of Holliday Junction Conformations

Holliday junctions are critical intermediates in DNA recombination, repair, and restart of blocked replication. Hexapeptides have been identified that bind to junctions and inhibit various junction-processing enzymes, and these peptides confer anti-microbial and anti-tumor properties. Earlier studies suggested that inhibition results from stabilization of peptide-bound Holliday junctions in the square planar conformation. Here, we use single molecule fluorescence resonance energy transfer (smFRET) and two model junctions, which are AT- or GC-rich at the branch points, to show that binding of the peptide KWWCRW induces a dynamic ensemble of junction conformations that differs from both the square planar and stacked X conformations. The specific features of the conformational distributions differ for the two peptide-bound junctions, but both junctions display greatly decreased Mg²⁺ dependence and increased conformational fluctuations. The smFRET results, complemented by gel mobility shift and small angle x-ray scattering analyses, reveal structural effects of peptides and highlight the sensitivity of smFRET for analyzing complex mixtures of DNA structures. The peptide-induced conformational dynamics suggest multiple stacking arrangements of aromatic amino acids with the nucleobases at the junction core. This conformational heterogeneity may inhibit DNA processing by increasing the population of inactive junction conformations, thereby preventing the binding of processing enzymes and/or resulting in their premature dissociation.     

DNA recombination: holliday junctions dynamics and branch migration

Holliday junctions are critical intermediates for homologous, site-specific recombination, DNA repair, and replication. A wealth of structural information is available for immobile four-way junctions, but the controversy on the mechanism of branch migration of Holliday junctions remains unsolved. Two models for the mechanism of branch migration were suggested. According to the early model of Alberts-Meselson-Sigal (Sigal, N., and Alberts, B. (1972) J. Mol. Biol. 71, 789-793 and Meselson, M. (1972) J. Mol. Biol. 71, 795-798), exchanging DNA strands around the junction remain parallel during branch migration. Kinetic studies of branch migration (Panyutin, I. G., and Hsieh, P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2021-2025) suggest an alternative model in which the junction adopts an extended conformation. We tested these models using a Holliday junction undergoing branch migration and time-lapse atomic force microscopy, an imaging technique capable of imaging DNA dynamics. The single molecule atomic force microscopy experiments performed in the presence and in the absence of divalent cations show that mobile Holliday junctions adopt an unfolded conformation during branch migration that is retained despite a broad range of motion in the arms of the junction. This conformation of the junction remains unchanged until strand separation. The data obtained support the model for branch migration having the extended conformation of the Holliday junction.