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

Abstract

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 NaBH 4 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 6 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 5Ј to an AP site, leaving 3Ј-hydroxyl and 5Ј-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 ␤-elimination by BER enzymes of another class, DNA glycosylases with an associated AP lyase activity (1, 9). In this case, an ␣,␤-unsaturated aldehyde is formed at the 3Ј-end, and a phosphate is formed at the 5Ј-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-1Ј 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 ␤-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 ␤-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 site...

Apurinic/apyrimidinic (AP) site recognition by the 5′-dRP/AP lyase in poly(ADP-ribose) polymerase-1 (PARP-1)

“…For example, human ribosomal protein S3 and nucleoside diphosphate kinase (NM23-H2/NDP) have been shown to form Schiff base intermediates with AP sites (15,16). In addition, the NaBH 4 trapping technique in combination with mass spectrometry has been used in a proteomic approach to identify a host of Escherichia coli and baker's yeast proteins reactive at AP sites (17); multiple proteins belonging to a range of metabolic pathways were identified. Because the AP site in DNA appears to be promiscuous in its binding to many cellular factors, it may be important for the BER system to sequester the AP site immediately upon formation.…”

Architecture and Metamorphosis

“…One is to regulate the cytoplasmic localization of MetRS and GluRS. Disruption of the complex by deletion of the GST-domain of Arc1p resulted in strong nuclear localization of all three components [78]; GluRS and Arc1p were found to associate with apurinic/apyrimidinic sites of damaged DNA in the nucleus [79]. In addition, the MetRS-Arc1p–GluRS complex is also important for GluRS trafficking into the mitochondria, which is important for providing an alternative pathway to the GlnRS activity that is lacking in yeast mitochondria [80, 81].…”

New Players in Recognition of Intact and Cleaved AP Sites: Implication in DNA Repair in Mammalian Cells

“…In general, search and identification of proteins reactive to AP sites include the following steps: (1) finding of cellular extract proteins forming covalent adducts with AP sites in DNA; (2) the design of AP-DNA probe containing a functional group providing the selective isolation of cross-linked DNA-protein products; (3) the preparative cross-linking of extract proteins with such DNA; (4) the affinity purification of the resulted products; (5) the identification of the protein in the covalent adduct with DNA using mass-spectrometry methods; (6) the confirmation of the results using purified proteins and/or specific antibodies and the known functions/interactions of the identified protein; and (7) the study of the functional role of the revealed interactions between proteins and AP DNA. A proofof-principle identification of protein reactive towards AP sites in species from E. coli and S. cerevisiae was reported in (Rieger et al, 2006). It should be noted that used protocol includes two steps that increases the selectivity of the method.…”

“…However, one cannot fully exclude the presence of impurity proteins with the same electrophoretic mobility as the target product. The identification was carried out for a number of proteins forming cross-links with AP sites in extracts of E. coli cells at logarithmic and stationary phases of cell growth (Rieger et al, 2006). It should be noted that authors used the previously developed non-enzymatic approach of the creation of AP sites in DNA based on the periodate oxidation of 2,3,5,6-tetrahydroxyhexyl phosphate precursor, which was introduced into an oligonucleotide during the standard phosphoroamidite oligonucleotide synthesis.…”

“…This method, in the authors' opinion, has several advantages over the more commonly used method that is based on the removal of uracil residues using uracil DNA glycosylase. The procaryotic proteins AroF, DnaK, MutM, PolA, TnaA, TufA, and UvrA from E. coli and eucaryotic ARC1 and Ygl245wp from yeast were identified (Rieger et al, 2006). Protein Ygl245wp with an unknown but essential for cell viability function was prepared in the individual state after its coding sequence was cloned, and the recombinant plasmid was used for the production of the protein in E. coli cells followed by its isolation.…”