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Originally published In Press as doi:10.1074/mcp.M500224-MCP200 on February 11, 2006.
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Molecular & Cellular Proteomics 5:858-867, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Proteomic Approach to Identification of Proteins Reactive for Abasic Sites in DNA *

Robert A. Rieger{ddagger}, Elena I. Zaika{ddagger},§,, Weiping Xie||, Francis Johnson{ddagger}, Arthur P. Grollman{ddagger},§, Charles R. Iden{ddagger},** and Dmitry O. Zharkov{ddagger}{ddagger},§§

From the {ddagger} Department of Pharmacology, § Laboratory of Chemical Biology, and || Proteomic Center, Stony Brook University, Stony Brook, New York 11794 and the {ddagger}{ddagger} Siberian Branch of the Russian Academy of Sciences Institute of Chemical Biology and Fundamental Medicine and Novosibirsk State University, Novosibirsk 630090, Russia


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Apurinic/apyrimidinic (AP) sites, a prominent type of DNA damage, are repaired through the base excision repair mechanism in both prokaryotes and eukaryotes and may interfere with many other cellular processes. A full repertoire of AP site-binding proteins in cells is presently unknown, preventing reliable assessment of harm inflicted by these ubiquitous lesions and of their involvement in the flux of DNA metabolism. We present a proteomics-based strategy for assembling at least a partial catalogue of proteins capable of binding AP sites in DNA. The general scheme relies on the sensitivity of many AP site-bound protein species to NaBH4 cross-linking. An affinity-tagged substrate is used to facilitate isolation of the cross-linked species, which are then separated and analyzed by mass spectrometry methods. We report identification of seven proteins from Escherichia coli (AroF, DnaK, MutM, PolA, TnaA, TufA, and UvrA) and two proteins from bakers’ yeast (ARC1 and Ygl245wp) reactive for AP sites in this system.

Apurinic/apyrimidinic (AP)1 sites are formed in DNA by spontaneous base loss and as a result of treatment with various mutagens (1). Excision of damaged DNA bases by DNA glycosylases during DNA repair results in AP site intermediates (1). Estimates of the rate of depurination in unstressed living cells vary from 0.05–0.15/106 bases/h obtained by extrapolation of rates of chemical depurination to physiological conditions (2, 3) to 10–30 lesions/106 bases/h (and the steady-state level of 5–20/106 bases in mammalian cells) measured by modification with a specific reagent in situ (4). Regardless of which estimate is more plausible, it is clear that AP sites represent a prominent type of DNA damage. AP sites may be unequally distributed over the genome (5), and their number increases greatly under challenging conditions such as x-ray irradiation (6). They are intrinsically unstable and can be converted to single strand breaks by heat, chemical bases, nucleophiles, or magnesium ions (1, 7). AP sites are highly mutagenic in vivo (8).

Normally AP sites are repaired through the base excision repair (BER) mechanism in both prokaryotes and eukaryotes. A group of dedicated enzymes, AP endonucleases, is responsible for safeguarding the genome against these lesions (1). AP endonucleases hydrolyze DNA 5' to an AP site, leaving 3'-hydroxyl and 5'-phosphate termini at the nick; such an intermediate is then processed through either short patch or long patch BER subpathways with the involvement of different DNA polymerases, DNA ligases, and accessory factors (9). AP sites can also be cleaved through ß-elimination by BER enzymes of another class, DNA glycosylases with an associated AP lyase activity (1, 9). In this case, an {alpha},ß-unsaturated aldehyde is formed at the 3'-end, and a phosphate is formed at the 5'-end of DNA at the nick that is further processed by AP endonuclease and the downstream BER enzymes. During AP site cleavage by AP lyase activity of DNA glycosylases, an aldimine intermediate is formed between an active site amine of the protein and the C-1' atom of the baseless deoxyribose (10, 11). This intermediate, usually referred to as a "Schiff base," is easily reduced with sodium borohydride or related compounds, forming a stable amino link between the enzyme and DNA; this reaction is widely used to prove the ß-elimination reaction mechanism for the enzymes capable of AP site cleavage (12). Some DNA glycosylases (e.g. Escherichia coli MutY protein) can form a Schiff base with no further ß-elimination (13), but their affinity for the AP site is still very high.

In addition to being a subject for recognition by specific DNA repair enzymes, AP sites interact, often unfavorably, with many other cellular processes. These lesions are known irreversible traps for mammalian topoisomerase I (14). DNA polymerases pause at AP sites followed by dissociation or highly mutagenic readthrough (15, 16); eukaryotic transcription also pauses at these sites (17). Tight binding to AP sites in DNA or the ability to be cross-linked to these lesions have been reported for a number of proteins including mammalian DNA polymerases ß and {gamma} and E. coli DNA polymerase I (18, 19); human immunodeficiency virus type 1 integrase (20); bacterial cytosine methyltransferases (21); several DNA ligases of phage and vertebrate origin (22); plant and bacterial ribosome-inactivating proteins (23–26); human and Drosophila S3 ribosomal protein (27, 28); and human nucleoside-diphosphate kinase NM23-H2/NDP (29). Thus, binding of cellular proteins to AP sites might be important for AP site repair or its protection from further degradation, might possibly serve for cell signaling/damage sensing, or, in other instances, might be an event interfering with normal protein functions. A full repertoire of AP site-binding proteins in cells is presently unknown, preventing reliable assessment of harm inflicted by these ubiquitous lesions and of their involvement in the flux of DNA metabolism.

To approach this problem, we devised a proteomics-based strategy for assembling at least a partial catalogue of proteins capable of binding AP sites in DNA. The general scheme relies on the sensitivity of many AP site-bound protein species to NaBH4 cross-linking. An affinity-tagged substrate is used to facilitate isolation of the cross-linked species, which are then separated and analyzed by mass spectrometry methods. Here we report a proof-of-principle identification of several protein species from E. coli and Saccharomyces cerevisiae reactive for AP sites and show that one of these, a yeast protein, Ygl245wp, of unknown function but required for viability, can be cross-linked to AP sites by sodium borohydride.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Oligonucleotides and Proteins—
Unmodified phosphoramidites and [1-N-(4,4'-dimethoxytrityl)-biotinyl-6-aminohexyl]-2-cyanoethyl-(N,N-diisopropyl)-phosphoramidite were purchased from Glen Research (Sterling, VA). 6-O-(4,4'-Dimethoxytrityl)-4-O-[(N,N-diisopropylamino)-(2-cyanoethoxy)phosphinyl]-1,2,5-O-tris(tert-butyldimethylsilyl)-3-deoxyhexitol was prepared as described previously (30). Oligonucleotide substrate for cross-linking in extracts, prepared by standard phosphoramidite chemistry, consisted of an HPLC-purified 30-mer, 5'-XGACGCTGAGAGTGTTTYGCCCTGAGACCG-3', annealed to a 25-mer, 5'-CGGTCTCAGGGCNAAACACTCTCAG-3', where X is biotin, Y is 3-deoxyhexitol, and N stands for A, C, G, T, or their equimolar mixture. To form the duplex, 80 µm 30-mer and 160 µm 25-mer were incubated in water for 2 min at 95 °C followed by 5 min at 37 °C and 30 min at room temperature. E. coli formamidopyrimidine-DNA glycosylase (Fpg) and Nei proteins were purified as described previously (31, 32). Purification of E. coli Nth will be described elsewhere. RNase-free DNase was purchased from Roche Applied Science. Lysozyme and protease inhibitor mixtures for use with bacterial and yeast cell extracts were from Sigma. Bacteriophage T4 polynucleotide kinase, Pfu Turbo DNA polymerase, NcoI and XhoI restriction endonucleases, and bacteriophage T4 DNA ligase were from New England Biolabs (Beverly, MA).

E. coli and Yeast Extract Preparation—
E. coli DH5{alpha} was plated onto LB agar from a frozen glycerol stock. After overnight incubation at 37 °C a single well defined colony was inoculated into 30 ml of LB and grown overnight at 37 °C in 250-ml conical flasks with shaking at 280 rpm. This culture (10 ml) was used to inoculate 1 liter of prewarmed LB in a 2.5-liter conical flask, and the growth was continued under the same conditions. The cultures were allowed to grow until midlog phase (A600 = 0.6–0.7, ~2 h) or for 24 h until saturation (A600 = 3–3.5). The cells were harvested by centrifugation (10,000 x g at 4 °C for 10 min), resuspended in 10 ml of TE buffer (pH 8.0)/g of cells, and frozen at –80 °C until needed. Immediately after thawing at 37 °C with intermittent shaking, the cell suspension was supplemented with protease inhibitor mixture (0.3 ml/g of cells) and lysozyme (50 µg/ml final concentration) and stirred for 30 min at 4 °C. Approximately 2 volumes of ice-cold TE buffer (pH 8.0) was added to reduce viscosity, and the suspension was centrifuged at 38,000 x g at 4 °C for 15 min. To the supernatant, crystalline ammonium sulfate was added to 80% saturation, and after a 1.5-h incubation on ice, the precipitate was collected by centrifugation (4,000 x g at 4 °C for 10 min). The pellet was dissolved in 10 ml of the cross-linking buffer (25 mm sodium phosphate (pH 6.8), 1 mm EDTA, 1 mm DTT, 10% glycerol), and the solution was dialyzed against two 1-liter changes of the same buffer (once overnight and once for 4 h).

Bakers’ yeast strain BY4743 was grown in yeast extract/peptone/dextrose medium supplemented with 100 µg/ml ampicillin at 30 °C with shaking at 240 rpm. An overnight culture was used to inoculate 1 liter of the medium (2 x 0.5 liters, 25 ml of inoculate/flask). When the culture reached A600 = 1, the cells (2.5 g) were harvested by centrifugation, washed once in phosphate-buffered saline, and frozen at –80 °C until needed. Thawed cells were resuspended in 10 ml of the cross-linking buffer supplemented with protease inhibitor mixture (50 µl/g of cells), and an equal volume of acid-washed glass beads (425–600 µm, Sigma) was added. The tube was vortexed vigorously for 30 s and transferred on ice for 30 s; 10 cycles of vortexing were carried out. The extract was clarified by centrifugation (17,000 x g at 4 °C for 10 min, repeated twice). The supernatant was batch-extracted twice with 1 ml of heparin-Sepharose (Sigma; room temperature, 15-min incubation, the sorbent pelleted by brief centrifugation). The sorbent was washed two times with 5 ml of the cross-linking buffer, transferred into an Ultrafree-CL centrifugal filter device (Millipore, Billerica, MA), and washed three times with 1 ml of the same buffer. Proteins bound to heparin were then eluted by washing the sorbent twice with 1 ml of the cross-linking buffer containing 1 m NaCl. The eluates were pooled and dialyzed overnight against 0.5 liters of the cross-linking buffer without NaCl. Both E. coli and yeast extracts were aliquoted and frozen at –80 °C. Protein concentrations were determined by Bradford assay.

Analytical Cross-linking—
Cross-linking of Fpg, Nei, and Nth for use as standards was performed as described earlier (31, 33) except for the different substrate sequence. For cell extracts, the reaction mixture (10 µl) contained the thawed extract (8 µl), 50 nm 32P-labeled oligonucleotide duplex, and 50 mm NaBH4. Cross-linking was performed for 30 min at 37 °C and terminated by adding 10 µl of gel loading dye (62.5 mm Tris-HCl (pH 6.8), 10% glycerol, 2% sodium dodecyl sulfate, 5% ß-mercaptoethanol, 12.5 µg/ml bromphenol blue). The sample was electrophoresed in a 12% discontinuous SDS-containing polyacrylamide gel (Laemmli system).

Preparative Cross-linking—
To remove nonspecific streptavidin-binding species, 10 ml of the thawed cell extract were treated with 0.5 ml of UltraLink immobilized streptavidin suspension (Pierce) for 30 min with gentle rocking at room temperature, and the streptavidin resin was removed by centrifugation (IEC clinical centrifuge, maximal speed at 4 °C for 3 min) repeated twice, leaving ~0.2 ml of the liquid over the pellet each time. Oligonucleotide duplex (3.6 nmol) was treated with 5 mm NaIO4 and added on ice to the cell extract in a flask large enough to accommodate the foam that forms vigorously during the subsequent reaction. Freshly dissolved NaBH4 (2 m) was quickly added to 50 mm final concentration, and the reaction mixture was transferred into a 37 °C water bath for 30 min. The liquid part of the reaction mixture was recovered and centrifuged to get rid of the residual foam.

Streptavidin Binding and Elution—
Immobilized streptavidin (250 µl) was added to the reaction mixture, which was then incubated as described above. The resin was centrifuged as above and washed three times by resuspension and centrifugation in 5 ml of the wash buffer (glycine-NaOH (pH 9.4), 2 m NaCl). The resin was then transferred into an Ultrafree-CL filter device, washed three times with 1 ml of the wash buffer and three times with 1 ml of water, and resuspended in 125 µl of water. 25 µl of DNase I reaction buffer stock (100 mm Tris-HCl (pH 7.5), 1.5 m NaCl, 10 mm MgCl2) and 100 units of RNase-free DNase I (Roche Applied Science) were added to the slurry, which was then incubated for 30 min at 37 °C with intermittent vortexing. The slurry was transferred into an Ultrafree-MC centrifugal filter device (Millipore) and centrifuged in a tabletop microcentrifuge for 1 min, and the liquid was dried under vacuum and dissolved in 10 µl of gel loading dye. The sample was resolved by 12% SDS-PAGE followed by staining with Coomassie Blue. Well defined protein bands were excised from the gel using a disposable surgical blade on a thoroughly cleaned glass surface and kept frozen at –20 °C until the analysis. The extract treated in the same way omitting the oligonucleotide duplex was used as a control.

Mass Spectrometric Analysis—
The techniques for identification of proteins by mass spectrometry have been reviewed thoroughly (34, 35), and methods for in-gel tryptic digestions have been developed (36, 37). Briefly, excised gel bands were destained by treating with 100 µl of 25 mm NH4HCO3 for 10 min and then adding an equal volume of CH3CN. After 20 min the solution was decanted, and 50 µl CH3CN were added. These steps were repeated until the stain was gone. Then the protein was reduced with DTT and alkylated using iodoacetamide under standard conditions (35). Gel slices were put into 50 µl of acetonitrile for 10 min; after removal of the solvent, 20 µl of trypsin (0.2 mg/ml) was added and permitted to react for 16 h at 37 °C. Peptides were extracted from the gel in 60% acetonitrile, 0.3% trifluoroacetic acid; dried to 5 µl; and purified on a ZipTip reverse phase column (Millipore). The column was washed six times with 0.1% TFA and eluted with 50% acetonitrile, 0.1% TFA. A 1:10 dilution of the final product was mixed with the MALDI matrix material ({alpha}-cyano-4-hydroxycinnamic acid), and 1 µl of the mixture was spotted at two locations on the MALDI target. An internal calibration mixture was added to one spot to improve mass accuracy; the second sample was externally calibrated on a similar mixture of peptides. The Voyager MALDI-TOF mass spectrometer (Applied Biosystems/MDS Sciex, Foster City, CA) was operated in the reflectron mode at 20-kV acceleration and 220-ns extraction delay. Spectra were obtained from an average of 100 laser shots over the m/z range 500–5000. Peptide mass mapping using the MASCOT software (Matrix Science, London, UK) was performed on a separate work station. The National Center for Biotechnology Information (NCBI) non-redundant protein database was interrogated using a mass tolerance of 100 ppm.

In selected cases samples were analyzed on the QSTAR Pulsar i LC/MS/MS instrument (Applied Biosystems/MDS Sciex) to verify the MALDI data analysis or to obtain sequence information on peptides from proteins not identified successfully by peptide mass mapping. The QSTAR mass spectrometer used an electrospray ion source and was interfaced to an LC Packings Nano LC system consisting of an Ultimate micropump, FAMOS microautosampler, and a Switchos microcolumn switch module (LC Packings, Sunnyvale, CA). Peptide separation was performed on an LC Packings PepMap nanocolumn (3 µm, C18, 75-µm inner diameterx 15 cm) using a 40-min linear gradient from 5 to 50% acetonitrile in 0.1% formic acid. Initially a survey scan was accumulated by the mass spectrometer over the mass range m/z 200–1600. Then CID spectra were acquired for 3 s over the range m/z 50–1600 using information-dependent acquisition MS/MS scanning. This data acquisition cycle was repeated over the course of the analysis. MASCOT software and the Swiss-Prot database were used for protein identification from the mass spectral data.

Cloning, Purification, and Cross-linking of Ygl245wp—
The coding sequence of Ygl245wp was PCR-amplified from 150 ng of yeast (strain BY4743) genomic DNA using Pfu Turbo DNA polymerase according to the manufacturer’s protocol. The primers used were 5'-CGAGACCCATGGGCACGAAACTATTTTCAAAGGTTAAGG-3' and 5'-CGAGACCTCGAGTTTCTTTGCACCATACTTGTTGACAG-3', carrying an NcoI and a XhoI restriction site, respectively. The product was treated with NcoI and XhoI and ligated into the pET-28a(+) vector (EMD Biosciences, San Diego, CA) at the respective restriction sites. Correctness of the insert was verified by direct sequencing at Stony Brook DNA Sequencing Facility. The resulting construct (pET-28a-Ygl245) codes for full-length Ygl245wp tagged with a hexahistidine oligopeptide at the C terminus. The recombinant plasmid was transformed into BL21(DE3) RIL E. coli cells (EMD Biosciences) by electroporation using an E. coli Pulser transformation apparatus (Bio-Rad).

To obtain recombinant Ygl245wp, 1 liter of 2x yeast extract/tryptone broth supplemented with 25 µg/ml kanamycin and 25 µg/ml chloramphenicol was inoculated with 20 ml of overnight culture of BL21(DE3) RIL E. coli carrying the pET-28a-Ygl245 plasmid and grown at 37 °C in an orbital shaker at 250 rpm. When A600 reached 0.5, the temperature was lowered to 25 °C, and after a 40-min adjustment period, the expression was induced by adding isopropyl 1-thio-ß-d-galactopyranoside to 0.2 mm (A600 = 0.85). After 5 h, the cells (4.5 g wet weight) were harvested by centrifugation and frozen until purification. After thawing at room temperature, the cells were resuspended in 40 ml of TE buffer (pH 8.0) supplemented with 1 mm phenylmethylsulfonyl fluoride and 1 mm DTT and stirred with 100 µg/ml lysozyme and 1 µg/ml DNase I for 20 min at room temperature. The incubation was continued at 4 °C for another 20 min after addition of NaCl to 1 m. The lysate was clarified by centrifugation (25,000 x g at 4 °C for 20 min), and 0.1 ml of 5% polyethyleneimine was added. After 1 h of stirring at 4 °C the solution was centrifuged as above, and the protein was precipitated by ammonium sulfate at 70% saturation. The pellet was dissolved in Buffer A (25 mm sodium phosphate (pH 7.5)) containing 500 mm NaCl and 50 mm imidazole and applied to a 5-ml HiTrap chelating column (Amersham Biosciences) charged with Ni2+. The hexahistidine-tagged protein was eluted by a 50–500 mm gradient of imidazole in Buffer A. The fractions containing a protein band of the expected size as judged by 12% SDS-PAGE were diluted 10-fold with Buffer A and applied to a 5-ml HiTrap heparin column (Pfizer) equilibrated in Buffer A with 100 mm NaCl. The column was developed with a gradient of 100–800 mm NaCl in Buffer A; the fractions of at least 95% purity (by Coomassie staining) were pooled; dialyzed against two changes of 250 ml of 25 mm sodium phosphate (pH 7.5), 400 mm NaCl, 1 mm EDTA, 1 mm DTT, 50% glycerol; and stored at –20 °C.

To assay cross-linking of purified Ygl245wp by NaBH4, the reaction mixture (10 µl) included 5 nm 32P-labeled oligonucleotide duplex and 150 nm Ygl245wp in 25 mm sodium phosphate (pH 6.8), 50 mm NaBH4 for 30 min at 37 °C. The products were resolved using 12% discontinuous SDS-PAGE


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Process Development—
We proposed a strategy to search for still unknown members of the list of proteins capable of interactions with AP sites. This strategy is based on a well known propensity of aldehydic baseless deoxyribose to react with amine moieties in its vicinity and on easy reduction of the resulting aldimine product by sodium borohydride. In this way, the protein bound to the AP site is irreversibly cross-linked to it. Thus, the full scheme of our experiments included the following: (i) preparation of cell extracts and oligonucleotides containing an AP site and an affinity tag, (ii) cross-linking in the extracts, (iii) affinity purification of the tagged cross-links, (iv) separation of affinity-purified products, (v) identification of the products by mass spectrometry, and (vi) verification of the results by cross-linking of individual purified proteins.

We carried out a set of experiments to find optimal conditions for removal of cell extract proteins nonspecifically bound to streptavidin matrix and for the most efficient recovery of cross-linked species bound to streptavidin through an oligonucleotide linker. Under neutral or moderately acidic pH, an unwanted protein background persisted even after washing the streptavidin beads at high ionic strength (data not shown). Only a combination of moderately alkaline pH (9.4) and stringent washing conditions (2 m NaCl) reduced the background binding in E. coli extracts to workable levels. Six washes under these conditions were sufficient to bring nonspecific adsorption close to zero while retaining constant levels of streptavidin-bound biotinylated oligonucleotide as judged by adsorption of a radioactively labeled probe (Fig. 1).


Figure 1
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  		FIG. 1. Efficiency of the washing protocol. The oligonucleotide duplex was 32P-labeled, incubated with E. coli extract (20 µl) in the absence of NaBH4, bound to streptavidin beads (20 µl), and washed with 6 x 50 µl of stringent washing buffer (see "Experimental Procedures"). Radioactivity in the supernatant ({circ}) and in pelleted streptavidin beads (•) was measured after each wash using a GSM-115 four-range survey meter (W. B. Johnson and Associates).

 
Streptavidin·biotin complexes are only disrupted under streptavidin-denaturing conditions. Although we have tested some of these conditions (guanidine chloride, urea, and SDS; data not shown) in an analytical mode, the denaturing agents would cause problems with PAGE separation when applied to preparative scale cross-linking. We therefore adopted nucleolytic degradation of the oligonucleotide linker as a way to specifically elute streptavidin-bound cross-linked proteins and tested the procedure on a model substrate, DNA cross-linked to E. coli Fpg protein, a known DNA glycosylase/AP lyase (9). Fig. 2 shows that DNase I efficiently degrades the DNA part of the Fpg·DNA cross-link; that Fpg, when cross-linked to a biotinylated oligonucleotide and bound to streptavidin, can be eluted by DNase treatment and a single centrifugation step; and that the presence of E. coli cell extract does not interfere with this procedure. The product of Fpg·DNA degradation by DNase I was heterogeneous, detected as two major bands on the gel, most likely due to the Fpg protein globule hindering DNase access to DNA proximal to the site of cross-linking.


Figure 2
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  		FIG. 2. Release of streptavidin-bound Fpg·DNA cross-link by DNase I. Lane 1, DNase I (1 µg); lane 2, Fpg (3 µg); lane 3, Fpg·DNA cross-link eluted from streptavidin by 1 µg of DNase I and centrifugation (Beads eluate I); lane 4, wash of the streptavidin beads with TE buffer after the centrifugation (Beads eluate II); lanes 5 and 6, same as lanes 3 and 4 but the Fpg·DNA cross-link was bound to streptavidin in the presence of E. coli cell extract; lane 7, Fpg·DNA cross-link treated with 0.1 µg of DNase I in solution; lane 8, DNase I after incubation with streptavidin beads and centrifugation; lane 9, Fpg·DNA cross-link prepared from 3 µg of Fpg and a 2-fold molar excess of the AP:C oligonucleotide duplex.

 
To show that known AP site-cross-linking proteins can be positively identified using this strategy, we prepared cross-links of three purified DNA glycosylases/AP lyases, E. coli Fpg, Nei, and Nth. These proteins were permitted to react with biotinylated DNA containing an abasic site in the presence of borohydride, and the complex was bound to streptavidin, released by DNase treatment, and separated by SDS-PAGE. Individual bands were excised, subjected to in-gel trypsinolysis, and analyzed on the Voyager MALDI-TOF instrument (Fig. 3). Data from the MS analysis were searched through the NBCI database using the MASCOT search algorithm. In the case of Fpg protein 36 mass peaks were used from the MALDI spectrum, mass tolerance was set at 100 ppm, and protein taxonomy was not limited. Results are shown in Table I; for each search the top hit identified the protein correctly, and the MASCOT confidence score for each search was at a high level. We estimated that 20 fmol of Fpg protein was used in the MS analysis, and approximately equal quantities were present in the analysis of the other proteins.


Figure 3
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  		FIG. 3. MALDI-TOF mass spectrum of an in-gel trypsin digest of a single band cut from a preparative gel. The MASCOT search algorithm assigned the Fpg protein with a confidence score of 93, matching 10 tryptic peptides with 44% sequence coverage and a mass accuracy of 7 ppm.

 

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  		TABLE I Identification of the proteins cross-linked to AP sites from standards

 
Cross-linking of Proteins to AP Sites in Cell Extracts—
In the NaBH4 cross-linking assay, DNA glycosylases with an associated AP lyase activity are the most reactive proteins presently known and (together with DNA polymerase ß) are the only ones for which formation of a Schiff base with an AP site has been proven relevant for biological function. Therefore, to estimate feasibility of the present cataloguing endeavor, we addressed the question of whether, and how many, proteins other than DNA glycosylases/AP lyases could be detected by borohydride cross-linking. Using a radioactively labeled duplex oligodeoxynucleotide containing an AP site, we showed that crude cellular extracts may be used to obtain a mixture of a number of proteins cross-linked to DNA (Fig. 4). A comparison of their mobility on gel electrophoresis with the mobility of E. coli Fpg protein cross-linked to a duplex of the same sequence shows that the molecular masses of cross-linked E. coli cell extract proteins vary widely from ~15 to 80 kDa. Fpg (molecular mass, 30.2 kDa) is typical of DNA glycosylases (the mass range of eight known E. coli DNA glycosylases is 18.7–39.1 kDa). This experiment showed that DNA bearing an AP site can be used as a universal substrate to sequester cross-linked species, many of which may represent unknown proteins. Because the oligonucleotide was labeled by polynucleotide kinase, only protein·DNA cross-linked complexes that contain intact 5' termini were observed, providing a reliable estimate of the yield of cross-linked species with an intact biotinylated 5' terminus. Different spectra of cross-linked species were observed in NaBH4-treated extracts of bacteria harvested in the exponential growth phase and in the saturated culture (Fig. 4), suggesting that the cellular fate of AP sites could strongly depend on the environmental conditions.


Figure 4
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  		FIG. 4. Storage phosphorimage of 5'-32P-labeled AP site-containing substrates after cross-linking in E. coli extracts and separation by 12% discontinuous SDS-PAGE. Lane 1, Fpg protein cross-linked to the substrate (size marker, 30.2-kDa protein part); lane 2, substrate incubated in the absence of the protein extract; lane 3, cross-linking with the extract prepared from a culture at the midlog phase of growth; lane 4, cross-linking with the extract prepared from a saturated culture.

 
We then attempted to perform large scale cross-linking and purification in the extracts of E. coli growing in either log or stationary phase. As a control, we used the same extracts and oligonucleotides incubated in the absence of NaBH4; purification on streptavidin resin was carried out in parallel following identical protocols. In both types of extracts, SDS-PAGE with Coomassie Blue staining revealed bands present in the NaBH4-treated preparation but absent from the control. The most easily discernible bands (two for the log phase extract, five for the stationary phase extract) were excised from the gel for mass spectroscopic analysis. In addition, bands corresponding to the mobility of DNase I and BSA (a stabilizing agent in the DNase I stock solution, Fig. 2) and pieces corresponding to blank parts of the gel were submitted for analysis.

In the same type of experiment, crude extracts of yeast cells produced a strong background of nonspecific binding that could not be eliminated even by washing under stringent conditions. Therefore, we batch-treated the yeast extracts with heparin-Sepharose, a sorbent for which many DNA- and RNA-binding proteins have affinity. The proteins adsorbed to heparin were eluted and used for cross-linking in the same way as were E. coli extracts (Fig. 5). Two NaBH4-specifc bands were excised and analyzed.


Figure 5
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  		FIG. 5. Coomassie Blue-stained gel of cross-linked products from an S. cerevisiae cell extract after purification on streptavidin beads, DNase elution, and separation by 12% discontinuous SDS-PAGE. Bands excised for analysis and their identifications are indicated. Lane 1, protein species recovered from streptavidin after cross-linking to the AP site-containing substrate; lane 2, protein species recovered after incubation in the absence of the substrate; lane 3, molecular mass markers (molecular masses indicated to the right).

 
A total of 18 gel slices from the experiments with E. coli were analyzed by MALDI-TOF and LC/MS/MS, including three gel blanks. DNase I and BSA, acting as internal positive controls, were found in one and three samples, respectively, whereas several slices did not yield a known protein. Positive identifications were made on seven proteins, and the results are shown in Table II. The MALDI-TOF peptide mass mapping technique identified eight proteins, six of which were verified on the QSTAR instrument using MS/MS techniques. In addition, the QSTAR identified four additional proteins not seen by the MALDI instrument but failed to find two proteins identified by the MALDI instrument. Two gel-bands were analyzed by MALDI-TOF from the yeast experiment, and a search of the mass spectral data identified two proteins (Table II).


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  		TABLE II Identification of the proteins cross-linked to AP sites in cell extracts

 
Cross-linking of Purified Ygl245wp—
Some of the proteins identified in the present screen (Fpg, DNA polymerase I, UvrA, and HSP-70) are already known to possess affinity for AP sites. Therefore, to provide evidence that our strategy can indeed identify hitherto unknown AP site-binding proteins, we cloned and purified the yeast Ygl245wp protein and studied its ability to be cross-linked to AP site-containing oligonucleotides in vitro. The biological function of this protein is presently unknown. It bears two conserved domains homologous to the catalytic domain and anticodon binding domain of glutamyl- and glutaminyl-tRNA synthetases (38); however, several RNA-interacting proteins have been shown to be active on AP sites in DNA (23–25, 27, 28).

We cloned Ygl245wp in a plasmid for expression in E. coli, purified the recombinant hexahistidine-tagged protein, and assayed its ability to be cross-linked to DNA. Fig. 6 illustrates the results of this experiment. A band with the mobility expected for a Ygl245wp cross-link was formed with AP sites positioned opposite any of the four canonical bases, although the reaction with the substrates containing pyrimidines opposite the AP site were slightly more efficient (Fig. 6, compare lanes 2 and 3 with lanes 4 and 5 and lanes 6 and 7 with lanes 8 and 9). The mobility and substrate preferences of the faster migrating band obtained on the gel suggest that it might come from low amounts of Fpg co-purified with Ygl245wp in our two-column system. If this is indeed the case, Ygl245wp cross-linking, albeit readily evident, should be rather inefficient. To check whether any low molecular weight compounds present in cells could improve cross-linking of Ygl245wp, we carried out the cross-linking reaction in the presence of cell extract filtered through a 3000-Da-cutoff membrane and found no difference with the reaction of Ygl245wp alone (Fig. 6, compare lanes 2–5 with lanes 6–9). We conclude that Ygl245wp can indeed be cross-linked to DNA containing an AP site through a Schiff base reduction mechanism.


Figure 6
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  		FIG. 6. Cross-linking of Ygl245wp to AP site-containing DNA duplex. Sample in lane 1 contained Fpg, lanes 2–9 contained Ygl245wp, and lane 10 contained no enzyme. The base opposite AP site in the duplex is indicated. Samples in lanes 6–9, in addition to Ygl245wp, contained 1 µl of cell extract filtered through a 3000-Da-cutoff membrane. Positions of Ygl245wp and Fpg cross-links are indicated by arrows.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
AP sites represent an important class of DNA lesions capable of interaction with a number of cellular proteins. Known examples of such interactions include several important DNA- and RNA-dependent proteins (1, 9, 10, 14–25, 27–29), and the list is very likely incomplete. Binding of proteins to AP sites in DNA may be important for DNA repair or protection as in the case of DNA glycosylases (1, 9, 10) or may be involved in deleterious effects of these lesions as with topoisomerase I (14). Our strategy to look for additional proteins reactive for AP sites was based on their ability to react with adjacent amine moieties with the product sensitive to NaBH4 treatment, generating a stable protein-DNA cross-link. In the present study we demonstrated the validity of the general scheme, which is likely to endure although its individual steps are doubtless open for technical improvement to increase yield and sensitivity.

An earlier work from our laboratory (30) describes a convenient way of postsynthetic introduction of aldehydic AP sites into oligonucleotides through incorporation of a 2,3,5,6-tetrahydroxyhexyl phosphate precursor and its conversion into a cyclic aldehyde by periodate oxidation. This work provided a very important advantage in our experiments over an often used method of AP site generation from dU by treatment with uracil-DNA glycosylase. This enzyme, although robust and easy to use, has increased affinity for its product, the AP site (39), and thus may prevent binding of some other proteins; procedures for removal of the enzyme are either cumbersome or potentially detrimental to the nascent AP site. In contrast, periodate chemistry allows full conversion of the precursor within minutes, and the oxidizing agent is easily quenched in situ by glycerol.

We used NaBH4 in cross-linking, which, in our experience, provides optimal reaction rates and product yields. Nevertheless other reagents for mild aldimine reduction, such as NaBH3CN (12) or alkylamine boranes (40), are also used in laboratory practice and may substitute for NaBH4 in reactions fine tuned for individual cross-linkable species.

The method of choice for affinity purification of cross-linked species was based on streptavidin-biotin interactions. Methods for synthetic introduction of biotin into oligonucleotides are well established (41), and many biotin affinity systems based on avidin or streptavidin are commercially available. Other tags also could be used, provided that they are stable under stringent washing conditions required to minimize background binding. One promising approach relies on forming polyacrylamide gel by co-polymerization of proteins cross-linked to duplexes bearing an acrylamide-tagged oligonucleotide (42) in a complementary strand followed by electrophoretic separation. Stripping the column of proteins of interest also affords room for improvement. Because the streptavidin-biotin interaction is extremely stable, we eluted the bound proteins by degrading the oligonucleotide linker by DNase. This approach is very efficient and reproducible but introduces easily identified but still undesirable additional protein species, DNase and BSA, to the mixture of cross-linked proteins. Some cross-linked species might have been missed due to the overlap in size with DNase or BSA. The next generation of the system may use a linker with a pre-engineered bond sensitive to certain compounds (e.g. a disulfide bond cleavable by thiol treatment) to allow mild chemical elution.

The cross-linked species in our experiments were separated by one-dimensional PAGE. Other separation methods, such as two-dimensional PAGE or chromatography, can be applied and could provide an advantage in separation of cross-linked proteins, possibly resolving species overlapping on a one-dimensional gel. In addition, more sensitive in-gel detection methods (silver or SYPRO staining) can be used to detect less abundant cross-linkable proteins.

Mass spectrometry has become the method of choice for protein identification, and the two proteomic techniques that were used in these experiments gave excellent results. Peptide mass mapping of tryptic fragments from gel-purified proteins using MALDI/TOF analysis is effective, and most procedures are automated. Although the sequence coverage of the identified tryptic fragments is often less than 50% of the protein, search algorithms give correct assignments based on the accuracy and precision of the m/z measurements. However, in cases where protein purification is less than ideal and multiple proteins are digested in a single gel slice, we have found that the peptide mass mapping technique may have more difficulty in assigning the correct protein because of the abundance of low intensity tryptic fragments in the MALDI mass spectrum. In such cases LC/MS/MS data acquired by the QSTAR instrument provides more accurate assignments because it is based on the partial sequence or the CID mass spectrum of a single peptide isolated by HPLC prior to analysis. A very high degree of specificity is achieved by the search algorithms, making this technique amenable to the analysis of more complex mixtures.

Finally a part of the overall scheme important to avoid false positives is an independent verification of cross-linking ability by demonstrating that the proteins identified by mass spectroscopy can indeed be cross-linked when expressed and purified. In E. coli extracts, we identified Fpg protein, a DNA glycosylase whose ability to be efficiently cross-linked to AP sites has been extensively documented (10, 12, 32, 43); indeed we used Fpg for process development and as a positive control in this study even before its identification as a species cross-linked in the extract. In addition, we showed that bacterially expressed and purified yeast Ygl245wp protein can be cross-linked to an AP site. The efficiency of this process was significantly lower than in the case of Fpg but comparable with those reactions reported for many other AP site-binding enzymes other than glycosylases (18–20, 22–25, 27, 29). Most likely, the highly efficient cross-linking of DNA glycosylases is due to formation of a Schiff base as a part of their reaction mechanism, whereas many other proteins (including monofunctional glycosylases) cross-link through an amino group adventitiously positioned near the aldehyde moiety of the baseless deoxyribose (13, 20) or possess low catalytic activity against intact AP sites, being more specific instead for some product of their further modification (44).

Ygl245wp is a protein required for yeast viability (45) with exact functions still unclear. Its homology to tRNA synthetases (38) suggests that it could be indispensable for protein synthesis. On the other hand, Ygl245wp has been shown to physically interact with yeast DNA repair/damage signaling proteins RAD51p (46) and MEC1p (47), and with subunit 4 of DNA replication/repair clamp loading protein replication factor C (47), thus raising the possibility that it could be involved in events associated with stalled replication forks (48). As discussed above, AP sites cause DNA polymerase to pause in vitro (15, 16) and have been shown to block replication forks at least in E. coli (49). Binding of Ygl245wp with RNA polymerase III (47) hints that interactions at the sites of stalled transcription are also possible, although the behavior of RNA polymerase III on AP sites has not been studied.

Our search also uncovered several proteins known to bind to AP sites with biological significance. In addition to a known DNA glycosylase/AP lyase Fpg, we trapped DNA polymerase I and UvrA nucleotide excision repair (NER) protein. DNA polymerase I is known to pause at AP sites (15, 16) and can be cross-linked to 5'-nicked AP sites (19), possibly participating in the repair of these lesions. UvrA, a central player in bacterial NER (1), has been shown to bind AP sites in the UvrABC excinuclease complex (50), indicating that NER could also play a backup role in the repair of AP sites.

Yeast Ygl245wp and E. coli elongation factor Tu identified as reactive for AP sites in cell extracts join the growing list of RNA-dependent proteins capable of interacting with AP sites in DNA, plant, and bacterial ribosome-inactivating toxins (ricin, gelonin, MAP30, and shiga toxins (23–26)) and S3 ribosomal protein (27, 28) among others. The significance of these interactions remains unclear, although they have been proposed to underlie an additional mechanism of toxicity of ribosome-inactivating proteins through damaging DNA (23–26) and provide backup pathways for repair of oxidative and abasic DNA lesions by S3 protein (27, 28, 51–55). Whether the reactivity of both known and newly identified RNA-dependent proteins toward AP sites is important for the biological activity of these proteins could be tested only by constructing the specific mutants deficient in AP site cross-linking but not in other known functions. The same applies to the proteins identified in the present screen and not known before as DNA-binding proteins (3-deoxy-d-arabinoheptulosonate-7-phosphate synthase and tryptophanase). Affinity of the former protein for AP sites could, in principle, arise from a certain resemblance of its substrate, 3-deoxy-d-arabinoheptulosonate-7-phosphate, to the 2-deoxy-d-ribose-5-phosphate structure of AP site.

Among the other proteins identified by us, HSP-70 of E. coli is of special interest. It has been shown recently that its human homologue binds to AP endonuclease and stimulates its activity by enhancing its binding to AP sites (56, 57). It is possible that HSP-70 can bind AP sites and thereby attract AP endonuclease to the lesion or can bind to the AP site together with AP endonuclease as a subunit of a functional DNA repair complex in cells. Because both human proteins are homologous to their E. coli functional counterparts, this interaction also could take place in bacteria.

The cross-linking method used in this study can potentially trap three classes of proteins: (i) true AP lyases that cross-link with DNA in a mechanism-based fashion, (ii) proteins that bind preferentially to AP sites in DNA and possess a free amino group positioned in the binding site close enough to react with C-1' of the baseless deoxyribose, and (iii) proteins with a similarly positioned amine function that bind any DNA. Of these, enzymes of the first class (DNA glycosylases/AP lyases) should be cross-linked most efficiently. E. coli possesses three such enzymes, Fpg, Nei, and Nth, of which we have so far found only Fpg among the species cross-linked in extracts. However, high reactivity of DNA glycosylases toward AP sites is counterbalanced by their low abundance. For example, Fpg is estimated to be present at ~100 molecules/cell (58), roughly 700-fold less than the amount of elongation factor Tu (59). Simple mass action considerations may explain why we identified only one known DNA glycosylase/AP lyase with the rest being more abundant but less reactive species. Some potentially reactive species could also have been disguised by DNase. Regarding Groups ii and iii, it could also be argued that the boundary between them is somewhat fuzzy. AP sites are well known points of greater DNA flexibility (60, 61), and it is being increasingly realized that higher flexibility of DNA generally facilitates its binding by proteins (62). Thus, even a protein usually binding DNA in a nonspecific mode could possess higher affinity for AP sites with possible biological significance; a recent study highlighting the importance of DNA flexibility for binding nonspecific DNA by human immunodeficiency virus type 1 integrase (63) fits nicely with the data on cross-linking of this enzyme to AP sites (20).

We used aldehydic AP sites within intact DNA for cross-linking. Some proteins could be better cross-linked to products of further AP site modification possessing an appropriate moiety, such as 5'-nicked AP sites (22, 44) or 2-deoxyribonolactone (64), which also could be used along the same general scheme with appropriate modification of cross-linking protocols. The outlined improvements in cross-linking, elution, staining, and analytical methods will likely increase the number of identified proteins reactive for AP sites.


   ACKNOWLEDGMENTS
 
We thank Dr. Shujuan Gao for providing yeast strains and yeast DNA, and we are grateful to Cecilia Torres and Maryann Wente for the preparation of all DNA oligomers.


  FOOTNOTES
 
Received, July 19, 2005, and in revised form, February 2, 2006.

Published, MCP Papers in Press, February 11, 2006, DOI 10.1074/mcp.M500224-MCP200

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 The abbreviations used are: AP, apurinic/apyrimidinic; BER, base excision repair; NER, nucleotide excision repair; TE, Tris-HCl/EDTA; Fpg, formamidopyrimidine-DNA glycosylase. Back

* This work was supported in part by National Institutes of Health Grants ES04068 and CA82902. Back

Present address: Dept. of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908. Back

§§ Supported by Wellcome Trust UK Grant 070244/Z/03/Z; Presidium of the Russian Academy of Sciences Program 10.5; Russian Foundation for Basic Research Grants 04-04-48253, 04-04-48254, and 05-04-48619; and United States Civilian Research and Development Foundation Grant Y2-B-08-08. To whom correspondence may be addressed. Tel.: 7-383-335-6226; Fax: 7-383-333-3677; E-mail: dzharkov{at}niboch.nsc.ru Back

** To whom correspondence may be addressed. Tel.: 631-632-8867; Fax: 631-632-7394; E-mail: charlie{at}pharm.sunysb.edu


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