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1 Pacific Northwest National Laboratory (PNNL), Molecular Biosciences Department, Richland, Washington 99352, USA
2 PNNL, Environmental Molecular Sciences Laboratory, Richland, Washington 99352, USA
3 Washington State University, School of Molecular Biosciences, Pullman, Washington 99164, USA
Reprint requests to: Eric Ackerman, Pacific Northwest National Laboratory, Molecular Biosciences Department, P.O. Box 999, Richland, WA 99352, USA; e-mail: eric.ackerman{at}pnl.gov; fax: 509-376-2149.
(RECEIVED January 2, 2001; FINAL REVISION March 27, 2001; ACCEPTED April 5, 2001)
4 Present address: Washington State University, School of Molecular Biosciences, Pullman, WA 99164, USA. 
Article and publication are at www.proteinscience.org/cgi/doi/10.1110/ps.40101.
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40–45 kDa) and gel filtration chromatography (92 kDa), as well as the consequences of DNA binding on its anomalous mobility. Iodoacetamide treatment of xXPA prior to SDS-PAGE yielded a single 42-kDa band, showing that covalent modification of Cys did not correct aberrant mobility. Determination of sulfhydryl content in xXPA with Ellman's reagent revealed that all nine Cys in active protein are reduced. Unexpectedly, structural constraints induced by intramolecular glutaraldehyde crosslinks in xXPA produced a 32-kDa monomer in closer agreement with its calculated molecular weight. To investigate whether binding to DNA alters xXPA's anomalous migration, we used gel filtration chromatography. For the first time, we purified stable complexes of xXPA and DNA ± cisplatin ± mismatches. xXPA showed at least 10-fold higher affinity for cisplatin DNA ± mismatches compared to undamaged DNA ± mismatches. In all cases, DNA binding did not correct xXPA's anomalous migration. To test predictions that a Glu-rich region (EEEEAEE) and/or disordered N- and C-terminal domains were responsible for xXPA's aberrant mobility, the molecular weights of partial proteolytic fragments from 5 to 25 kDa separated by reverse-phase HPLC and precisely determined by ESI-FTICR MS were correlated with their migration on SDS-PAGE. Every partial tryptic fragment analyzed within this size range exhibited 10%–50% larger molecular weights than expected. Thus, both the disordered domains and the Glu-rich region in xXPA are primarily responsible for the aberrant mobility phenomena. Keywords: XPA; ESI-FTICR mass spectrometry; DNA repair; gel electrophoresis; SDS-PAGE; intrinsic disorder; partial proteolysis
Abbreviations: cisplatin, Cis-diamminedichloro-platinum(II) • EMSA, electrophoretic mobility shift assay • ESI-FTICR MS, electrospray ionization interface coupled to a Fourier transform ion cyclotron resonance mass spectrometry • XPA, Xeroderma pigmentosum group A
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Introduction |
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We recently performed time-resolved trypsin proteolysis on active, full-length recombinant Xenopus XPA protein (xXPA) to better characterize its structural features (Iakoucheva et al. 2001). Partial proteolytic fragments were analyzed by electrospray ionization interface coupled to Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry (MS) and a novel algorithm designed to predict disorder in proteins. Our results were consistent with the interpretation that about two-thirds of the molecule is flexible or disordered, mostly at the N and C termini. The remainder constitutes an ordered core containing a Zn-finger and approximately corresponding to the minimal binding domain (MBD) of human XPA (hXPA), whose NMR structure is known (Buchko et al. 1998; Ikegami et al. 1998).
Aberrant mobility of XPA under denaturing conditions was reported earlier but not studied in detail. Multiple bands of nearly equal intensity exhibiting anomalously high mobility on SDS-PAGE under reducing conditions in the Laemmli system (Laemmli 1970) were previously observed for hXPA and some of its deletion mutants (Miyamoto et al. 1992; Kuraoka et al. 1996). Here we investigate these migration phenomena for xXPA using gel filtration chromatography, ESI-FTICR MS, and SDS-PAGE. Despite some sequence differences between the human and Xenopus protein (67% amino acid identity), migration abnormalities for XPA are conserved between species. Our objective was to test whether multiple bands and aberrant mobility were caused by a single parameter such as reduced/oxidized sulfhydryls, a highly charged Glu-rich region, the long disordered domains, or a combination thereof. The extreme precision provided by ESI-FTICR MS greatly facilitated assignment of xXPA's fragments to their sequences and mobility. A question that needs to be addressed is whether these parameters correlate with putative structural changes in XPA that may have direct functional consequences in DNA repair.
Disorder-to-order transitions upon DNA or protein binding facilitate shape accommodations so that proteins with significant disordered regions could bind to a wide variety of structurally distinct substrates (Kriwacki et al. 1996; Wright and Dyson 1999). This explanation is consistent with both xXPA's disordered domains and its function to recognize and bind to many different bulky adducts, as well as interact with other DNA repair proteins. Previous reports that used electrophoretic mobility shift assays (EMSA) (Jones and Wood 1993; Lao et al. 2000) or filter-binding assays (Asahina et al. 1994) to study XPA binding to DNA ± various lesions showed a surprisingly low preference for damaged over undamaged DNA despite the high fidelity of NER. Thus, an additional objective of our study was to test whether gel filtration chromatography might complement EMSA and perhaps detect higher preference of xXPA for damaged DNA. First we showed that xXPA–DNA complexes were stable during gel filtration chromatography. We then determined whether xXPA binding to DNA ± lesions caused conformational changes that significantly altered its aberrant migration under native conditions. Finally, we discussed the possible causes of xXPA's migration phenomena.
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Results and Discussion |
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) contains no post-translational modifications or any putative disulfides because its molecular weight determined by ESI-FTICR mass spectrometry (30922.02 Da) agreed within 0.43 Da to that calculated from the sequence (30922.45 Da). Nonetheless, its SDS-PAGE mobility was 42 kDa, 40% larger than expected (Fig. 2A). Furthermore, xXPA usually migrates as 2–4 bands of nearly equal intensity ranging in size from 40–45 kDa (Fig. 2A, lane 1). Interactions with Zn were eliminated as a factor in both anomalous migration and multiple bands by pretreatment of xXPA with 100 mM EDTA prior to electrophoresis (not shown). One possibility to explain the anomalous migration and the multiple bands might be incomplete reduction of some or all of the nine Cys in xXPA (Fig. 1), even though fresh 5 mM DTT was present in all buffers during purification and 100 mM DTT was used in the SDS-PAGE loading buffer. To test whether the sulfhydryls are indeed in the reduced state prior to gel electrophoresis, we estimated free sulfhydryls using Ellman's reagent (Fig 2B). These data clearly show that all nine cysteines in xXPA are reduced under the conditions used to purify this protein. Lack of disulfides in xXPA is also consistent with our ESI-FTICR MS assignment, which precisely matched its expected mass.
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To covalently modify xXPA's reduced SH groups (Hermanson 1996) and thereby prevent disulfide bonds formation during the electrophoresis, xXPA was alkylated with iodoacetamide. This treatment eliminated the multiple bands between 40–45 kDa (Fig 2A
, lane 1), yielding a single band of 42 kDa (Fig 2A, lane 2), which remains far above xXPA's molecular weight. Thus, the sulfhydryl groups contribute to the alternative bands, but blocking them does not correct the aberrant mobility.
The calculated and SDS-PAGE-determined molecular weights for most unmodified proteins agree within ±5%, presumably due to charge and shape compensation involving differential SDS binding (Dunker and Rueckert 1969). The 40% divergence between calculated and observed sizes for xXPA falls several standard deviations outside the norm and it is consistent with other examples for anomalous migration (e.g., Klenova et al. 1997). Aberrant migration on SDS-PAGE for recombinant fibrinogen fragments sometimes exceeds 400% due to a high incidence of an unusual 
-helical coiled-coil structure (Query et al. 1989). For other proteins, abnormal mobility (
160%) sometimes results from localized regions of high charge (Query et al. 1989). xXPA's Glu-rich region (EEEEAEE) between 70–76 therefore might be a potential explanation for aberrant mobility.
ESI-FTICR MS identification of aberrantly migrating fragments
A protein's migration anomaly might be associated with one or just a few local sequence regions containing excess positive or negative charge. If so, one or a few peptide fragments would exhibit anomalous mobility, whereas the others would exhibit the usual mass/migration relationship. To evaluate whether particular regions in xXPA were responsible for its aberrant mobility, partial proteolysis fragments covering 100% of the protein sequence were separated by reverse-phase HPLC and then analyzed by both SDS-PAGE and ESI-FTICR mass spectrometry (Fig. 3, Table 1). SDS-PAGE lacks sufficient resolution to identify discrete proteolysis fragments, especially those of similar size. ESI-FTICR MS provides precise mass determinations of individual fragments within complex mixtures and deduction of the sequence corresponding to each fragment, even for polypeptides not well separated by SDS-PAGE.
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10 bands larger than 14 kDa and presumably many low molecular weight fragments (<14 kDa) were likely present, only two or three total peaks were found using several reverse-phase protocols (not shown). Nonresolving reverse-phase chromatography conditions included solubilizing samples in 6M guanidine-HCl (or 8M urea) ± 40–120 mM DTT followed by MeCN gradients in 0.1% TFA. Alternate ion pairs for TFA with increasing MeCN gradients, such as 10 mM ammonium acetate at pH 8.0 or 10 mM methylphosphonic acid at pH 6.5, 100 mM sodium perchlorate also failed to improve resolution. The most effective chromatography separations yielded only three to five well-resolved peaks, each of which contained multiple proteolysis fragments when the recovered fractions were analyzed by SDS-PAGE (Fig. 3, Table 1). Similarly poor resolution of partial proteolysis products with reverse-phase HPLC has been reported for other proteins, for example, stathmin (Redeker et al. 2000). Tryptic fragments separated by reverse-phase HPLC are listed in order of decreasing molecular weight in Table 1. Fourteen additional fragments below 4 kDa identified by ESI-FTICR MS were in the nonlinear range of the SDS-PAGE resolution (not shown). For each fragment, the ESI-FTICR mass spectrometry measured and calculated molecular weights agree extremely well. In contrast, the molecular weight estimated by SDS-PAGE diverged 11%–53% from the calculated molecular weight. Every analyzed fragment between 5–25 kDa revealed aberrant mobility, suggesting that the origin of the anomalous migration of xXPA is not localized to just one region.
Full-length xXPA's 40% deviation from the expected migration on SDS-PAGE becomes 30% for fragments without the first 40 N-terminal and the last 60 C-terminal amino acids (Table 1). This deviation falls even further to 15% for fragments confined to the ordered core (85 to 200) lacking the highly charged E70-E76 region. Moreover, the ordered core region in hXPA that comprises the minimal DNA-binding domain also migrates about 15% larger than expected (not shown). The xXPA C-terminal fragment exhibits the largest mobility difference between molecular weight on SDS-PAGE and that calculated from the sequence. This result is consistent with our findings that disordered regions display higher discrepancy in SDS-PAGE mobility. We conclude the cause for xXPA's aberrant mobility correlates primarily with both the highly charged Glu-rich region and with its large disordered terminal domains.
Glutaraldehyde crosslinking demonstrates intra- and intermolecular crosslinks
Modification of amines with glutaraldehyde results in both intra-and intermolecular crosslinks (Hermanson 1996). Addition of glutaraldehyde to ovalbumin control at 0.05% (v/v) did not change its SDS-PAGE mobility, yet the same glutaraldehyde concentration produced numerous apparent inter- (*) and intramolecular (
) crosslinks in xXPA (Fig. 4). The gel shows more putative intermolecular crosslinks at lower glutaraldehyde concentrations (Fig. 4, lanes 6–7), perhaps because some intermolecular crosslinked oligomers failed to enter the gel. At 0.05% glutaraldehyde, most of the xXPA aggregated at the top of the gel. The discrete patterns of intramolecular crosslinked products indicate there are preferred sites for glutaraldehyde reactivity. Precise identification of these sites would yield structural insights into full-length XPA, whose known structure is limited to only a minimal DNA-binding domain (Buchko et al. 1998; Ikegami et al. 1998). Interestingly, the intramolecular crosslinks produced a monomeric xXPA that migrated closer to its expected molecular weight of 31 kDa. Denaturing with SDS generates an open, unconstrained protein structure that generally provides accurate mass assignment on SDS-PAGE. Intramolecular crosslinking constrains protein structure, thereby preventing complete unfolding by SDS and would be expected to yield inaccurate mass assignments by SDS-PAGE. Our results show that intramolecular crosslinking produced a more accurate assessment of xXPA's molecular weight by SDS-PAGE, that is, the exact opposite of the expected result and the first reported example for glutaraldehyde correcting an anomalous molecular weight. The more intramolecular crosslinks at higher glutaraldehyde concentrations, the closer xXPA migrates to its expected mobility (Fig. 4, lane 4 vs. 7). Up to 10-fold lower concentrations of glutaraldehyde yielded bands consistent with xXPA oligomers, although future experiments would be needed to identify these species.
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The crosslinking results on xXPA can be evaluated in terms of current models for protein mobility in SDS-PAGE. The necklace model for SDS-protein complexes was based on studies showing that, in the absence of gel, SDS-protein complexes migrate irrespective of their size. This model, in which micelles are distributed along the partially folded peptide chain (Shirahama et al. 1974) was confirmed by direct visualization using cryo-electron microscopy (Samso et al. 1995). The sieving effects of gels presumably facilitate the separation of proteins according to their size. Combining the necklace and sieving models (Westerhuis et al. 2000) can describe the electrophoretic behavior of the SDS-protein complexes on gels. The increased mobility of xXPA following glutaraldehyde crosslinking can be explained by these electrophoretic models assuming that the extended, intrinsically disordered regions of xXPA are not covered by SDS micelles, as are the polypeptide chains of globular proteins. Because intrinsically unstructured protein regions lack hydrophobic clusters sufficient to induce folding, they might also be unable to nucleate SDS micelle formation. Thus the collapse caused by glutaraldehyde crosslinking is not compensated by reduced SDS binding. These conjectures to explain xXPA's aberrant electrophoretic mobility suggest that proteins with intrinsically unstructured regions of sufficient size would frequently migrate anomalously in SDS-PAGE.
Aberrant behavior on gel filtration chromatography
The molecular parameters influencing apparent molecular weight on gel filtration and SDS-PAGE are size, shape, and charge of the molecule (Hollecker 1997). Correlating molecular weight into a measure of size is influenced by hydration, partial specific volume, and protein geometry (e.g., flexible or random coils, spheroid, ellipsoid, etc.). The charge density and free mobility depend on the amount of bound SDS (Poduslo and Rodbard 1980), which preferentially interacts with basic or hydrophobic amino acids. The overall composition of xXPA reflects comparable percentages of nonpolar, polar, acidic, and basic amino acids. Furthermore, the composition of disordered N and C termini and the ordered core do not significantly differ from that of the full-length molecule (Table 2). Thus the overall charge and hydrophobicity of xXPA do not necessarily imply unusual SDS mobility, with the exception of the E70–E76 region.
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). Gel filtration chromatography is moderately accurate if the protein is roughly the same shape as the standards used to calibrate the column (Deutscher 1990). Aggregation of samples on the column is an unlikely cause for the 300% higher apparent molecular weight than expected because there is a linear relationship between log molecular weight and Kav (Fig. 5A) for the protein standards and a very symmetrical shape for the xXPA elution profile (Fig. 5B). It is also unlikely that xXPA's aberrantly large size estimated by gel filtration chromatography is due to binding the column matrix because the column-independent Kav is <1; this is a good indicator that interaction with the matrix is minimal (Deutscher 1990). Furthermore, the same molecular weight was observed at different flow rates and in a different buffer (see Materials and Methods), as well as when using a different matrix, i.e., Pharmacia Superdex 200 (not shown) rather than Pharmacia Superose 12. We conclude that xXPA under native conditions has an unusually extended shape, which can contribute to its aberrant behavior during gel filtration chromatography. An abnormally high apparent molecular weight by gel exclusion has been used as an indicator for intrinsically unfolded protein (Kriwacki et al. 1997).
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, 
) or cisplatin DNA (Fig. 6B, 
) were sufficiently stable to withstand gel filtration chromatography. Previous reports showed XPA has only a two- to threefold preference for damaged DNA (Jones and Wood 1993; Asahina et al. 1994; Lao et al. 2000). Our data demonstrates significantly more complex (at least 10-fold) forms with the cisplatin substrate as shown by disappearance of the free DNA peak from the chromatographic profiles (Fig. 6A,B, • vs. 
) and by densitometric analysis of an electrophoretic mobility shift assay (EMSA; Fig. 6C) of chromatographic fractions. This analysis confirms peak assignments and indicates good recovery of both DNA and protein after chromatography. Nearly all (80%) of the cisplatin DNA is present in the complex, whereas only 5% of the undamaged DNA is bound to xXPA. This means that xXPA–(cisplatin) DNA complex either forms more readily and/or is more stable to gel filtration chromatography than the xXPA–(undamaged) DNA complex, as expected for a DNA-damage-recognition protein. Thus xXPA exhibits at least two DNA-binding modes and DNA with damage is preferred to undamaged DNA. Our results indicate that gel filtration chromatography is a sensitive assay to investigate xXPA–DNA interactions.
Previous studies showed that enhanced NER occurred at sites that contained both cisplatin adducts and mismatches (Moggs et al. 1997) or both cyclobutane pyrimidine dimers and mismatches (Mu et al. 1997). However, distortion to DNA without a covalent modification is insufficient for NER because small loops and mismatches are repaired very poorly, if at all (Hess et al. 1997; Moggs et al. 1997; Mu et al. 1997); although one recent EMSA analysis showed preferential binding of hXPA to 19-mer DNA containing three mismatches (Buschta-Hedayat et al. 1999). Consistent with the NER studies, our gel filtration results show that xXPA can not discriminate between undamaged DNA and DNA with two to four mismatches (Fig. 6C, lanes 1–6). Furthermore, xXPA can not discriminate between cisplatin DNA with mismatches and cisplatin DNA without mismatches (Fig. 6C, lanes 7–12). Complexes containing DNA with TT mismatches opposite the cisplatin-GG, or TTTT mismatches opposite the T(cisplatin)GGT showed about the same amount of bound DNA that was still 10 times higher than in complexes with undamaged DNA ± mismatches.
Next we investigated whether xXPA binding to DNA significantly alters its apparent molecular weight. Binding to DNA ± damage results in a single symmetrical peak. The elution position of the complex corresponds to 100 kD (Fig. 5A), which is about three times its expected size. Thus, DNA binding does not cause a conformational change that significantly alters xXPA's 92-kD anomalous migration on gel filtration chromatography.
Is there a correlation between our disorder/order data and known mutations in XP patients? There are several known mutations in introns and missense mutations affecting hXPA splice sites, but nearly all reported coding-region point mutations are located in the ordered minimal DNA binding domain, especially in the Zn-finger region. The nearest N-terminal mutation (L94 to P94) is at the boundary between the disordered domain and the ordered MBD. Nonmissense, coding-region mutations on the C-terminal side of the MBD include only a single reported substitution, H244 to R244 (States et al. 1998). Thus the clinical cases reveal that amino acid mutations in both the intrinsically disordered N- and C-terminal domains occur rarely, if ever, while point mutations, insertions, deletions, and missense mutations are clustered in the ordered domain. Further analyses of mutation rates in ordered versus disordered regions are needed to determine the significance of this observation.
In summary, both native xXPA ± DNA on gel filtration chromatography and xXPA on SDS-PAGE exhibit aberrantly large migration behavior. The causes for this phenomena, as determined by SDS-PAGE, are not restricted only to the highly charged Glu-rich region, but are also related to the protein's unusual shape derived mainly from its disordered N- and C-terminal domains. These disordered regions are highly conserved in five species (Iakoucheva et al. 2001) and probably relate to XPA's functional roles in DNA damage recognition and repair. Intramolecular crosslinks at defined locations partially correct xXPA's aberrant mobility. Identifying crosslink sites in proteins and complexes by ESI-FTICR MS will provide a new assay to probe interactions, particularly for disordered domains that are difficult to study by NMR or crystallography. This approach could complement site-specific hydrogen exchange studies to map disorder and conformational changes (Nettleton and Robinson 1999). DNA binding does not significantly change xXPA's anomalous behavior on gel filtration chromatography under native conditions. However, gel filtration revealed greater preference by xXPA for DNA-containing lesions than previous studies. Further experiments with additional repair proteins ± DNA may be necessary to reveal conformational changes that correct its migration.
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Materials and methods |
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Ioadacetamide reaction
After xXPA purification and concentration, freshly prepared DTT was added to a final concentration of 10 mM and incubated at 50°C for 10 min. After cooling to room temperature, iodoacetamide was added to a final concentration of 25 mM, incubated for 15 min in the dark at room temperature, and loaded on 10% SDS-PAGE in the SDS-loading buffer containing fresh 100 mM DTT.
Reverse-phase chromatography
Limited proteolysis of xXPA, analysis by ESI-FTICR and SDS-PAGE were as previously described (Iakoucheva et al. 2001). After limited proteolysis, the fragments were solubilized by the addition of guanidine HCl to 6 M, and DTT to 120 mM; 100 µL samples were boiled 3 min; then 400 µL 6% acetonitrile/0.1% trifluoroacetic acid was added immediately and samples were loaded directly onto a Vydac C4 214MS5215 column equilibrated in 0.1% TFA and 5% MeCN. The column was washed with 10-column volumes (CV) of 5% MeCN to remove excess salts and then consecutive gradients of 5–15% MeCN (1.5 CV) at 2.5%/min, 15–42.5% MeCN (11 CV) at 1%/min, and 42.5–55% acetonitrile (2 CV) at 2.5%/min were run. All column buffers contained 0.1% TFA. Fractions were taken at 1 min intervals and aliquots were analyzed by SDS-PAGE and by ESI-FTICR mass spectrometry. This approach allowed a better comparison of the SDS-PAGE mobility with the measured masses of the fragments since aliquots of the same fraction were used in both experiments.
Electrophoresis
Limited proteolysis reactions were terminated by boiling 5 min in SDS-PAGE loading buffer containing 100 mM DTT. The 10% and 16% Tris-glycine gels were prepared as described (Laemmli 1970) and all other gels were purchased from Novex.
Determination of sulfhydryl content with Ellman's reagent
Ellman's reagent and Cysteine standards were purchased from Pierce and used according to the manufacturer's instructions. The concentration of purified recombinant xXPA was determined using the molar extinction coefficient of 29,940 calculated according to Mach et al. (1992). The sulfhydryl concentration in xXPA was determined from the calibration curve obtained for the Cysteine standards.
Gel-filtration chromatography
Gel-filtration Superose 12 HR 10/30 column was purchased from Pharmacia. The column was equilibrated overnight in 0.5M NH4OAc at pH 7.5 (Buffer A). Similar results were obtained when using the same column in 25mM HEPES-KOH at pH 7.5, 200mM KCl, 5mM MgCl2, 5mM DTT (Buffer B). For accurate determination of xXPA molecular weight, the High and Low Molecular Weight Gel Filtration Calibration Kits (Pharmacia) were used for preparation of the calibration curve, as described by the manufacturer. The elution volumes of the standards were determined and then xXPA (250 µg) was loaded. Kav for xXPA was calculated using formula 
, where Ve, elution volume for the protein; V0, column void volume; Vt, total bed volume. xXPA MW was estimated from the calibration curve. For forming xXPA–DNA complexes the binding of 120 µg xXPA to 5 µg DNA was performed in (Buffer B), the mixture was incubated for 30 min at 30°C and loaded on the gel filtration column.
Glutaraldehyde crosslinking
Twenty-five percent aqueous solution of glutaraldehyde was purchased from Sigma. The concentrated xXPA was dissolved in 10 mM NaPi at pH 7.5, 150mM NaCl (30 µL reaction volume, 5 µg of xXPA per reaction) and glutaraldehyde was added to final concentrations of 0.05%, 0.02%, 0.01%, and 0.005%. The mixture was incubated for 30 min at 25°C, the SDS-loading buffer containing 100 mM DTT was added and the samples were boiled 3 min and loaded on the gradient 4%–20% SDS-PAGE.
N-terminal sequencing
Limited proteolysis reactions were electrophoresed on glycine-free SDS-PAGE, 4%–12% Bis-Tris gel (Novex) and electroblotted onto PVDF membrane (Moos 1998). N-terminal sequencing was done in the Laboratory for Bioanalysis and Biotechnology, Unit 1, at Washington State University on an Applied Biosystem 475A according to manufacturer's instructions.
DNA substrates and EMSA
Oligonucleotide used for the XPA-DNA binding studies were as follows:
ESI-FTICR MS confirmed a single cisplatin in each oligonucleotide (Xu et al. 1999). Binding conditions in Buffer B were as described for gel filtration, and the peaks corresponding to xXPA–DNA complexes and free DNA were collected and electrophoresed on 8% native polyacrylamide gel in 0.5 X TAE with addition of 0.5mM MgCl2 (both in gel and running buffers) at 10V/cm for 2 h in the cold (4°C). The bands were visualized by staining the gel with SYBR Green (Molecular Probes) according to manufacturer's instructions. The amount of DNA in each band was quantitated using NIH Image.
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