Selection and identification of proteins bound to DNA triple-helical structures by combination of 2D-electrophoresis and MALDI-TOF mass spectrometry

Abstract

Identification of proteins binding specifically to peculiar nucleic acid structures can lead to comprehension of their role in vivo and contribute to the discovery of structure-related gene regulation. This work was devoted to establishing a reliable procedure to select proteins on the basis of their interaction with a nucleic acid probe chosen to fold into a given structure. 2D-electrophoresis and mass spectrometry were combined for protein identification. We applied this procedure to select and identify triplex-binding activities in HeLa nuclear extracts. To achieve this, we used a panel of deoxyribonucleic probes adopting intramolecular triple-helices, varying in their primary sequence, structure or triple-helix motif. A limited number of spots was reproducibly revealed by South-western blotting. Spots of interest were localised among a complex population of (35)S-labelled proteins according to their (32)P-specific emission. Position of the same spots was extrapolated on a preparative gel coloured with Coomassie blue, allowing excision and purification of the corresponding proteins. The material was subjected to mass spectrometry upon trypsin digestion and MALDI-TOF peptide fingerprinting was used for research in databases: five of them were identified and found to belong to the hnRNP family (K, L, A2/B1, E1 and I). The identities of several of them were confirmed by comparing western and South-western blots on the same membrane using specific antibodies. The recognition specificity of most of these proteins is large, according to previous reports and our own experiments. It includes pyrimidine-rich DNA sequences in different contexts: single strand to a small extent, triplex and possibly other higher-order structures.

Figure 1

Sequences and structures of the DNA probes. 55mers adopting intramolecular triplex structures are represented. The letters T, C, D, S stand for Triplex, related Controls of less stability, Duplex and Single strand, respectively. Indices (1, 2, 3) stand for different nucleotidic compositions in the third strand. (T, D, S) and (T′, D′, S′) are used for distinct duplex motifs. (op) and (np) stand for the nature of phosphodiester and phosphoramidate backbone, respectively. A variation was introduced for T1 probe, replacing T4 and T6 loops by tri-ethylene glycol T1(teg).

Figure 2

Gel retardation with 55mer intramolecular triplexes. Lanes 1–4: in the presence of 2.5 µg of nuclear HeLa extract; lanes 5–8: without extract. Lanes 1, 2, 5 and 6: T1′; lanes 3, 4, 7 and 8: T1 lanes 1, 3, 5 and 7: the third strand has a np backbone; lanes 2, 4, 6 and 8: the third strand is op. D.BP and T.BP stand for duplex and triplex-binding proteins, respectively, as previously shown by competition experiments (5). 55mer designs a bi-molecular species where the hairpins are probably paired.

Figure 3

South-western blot of 1D-protein gels in the presence of intramolecular or intermolecular triplexes. Intramolecular 55mers were 5′-labelled with T1 and T2 (op) containing either a op third strand (lanes 1 and 2, respectively), or a np third strand T1(np) (lanes 3 and 5). Lanes 3, 4 and C were made with radiolabelled nuclear extracts, lane C was not hybridised. The control formed with labelled S1(np) is shown in lane 4. Experiments were made in the presence of different third strand motifs and the corresponding unlabelled competitors: T2, (CBrdU): lanes 6, 7 and 8; T3′, (TCG) lanes 9, 10 and 11; T1′ (TmC) lanes 12, 13 and 14, and controls obtained with the third strand alone (S3′: lane 16) and S1 (lane 15). Competition experiments were performed in the presence of a 100-fold excess (20 µM) of cold T (lanes 7, 10 and 13), or cold S (lanes 8, 11 and 14). South-western blot was performed with intermolecular triplexes formed between S1 (np) and 5′-labelled D or 5′-labelled S1 (np) and D and was compared with intramolecular triplex T2 (lanes 17, 18 and 19, respectively). The proteins are designed by bold arabic letters chosen as a function of the name of the corresponding protein(s) whenever identified. The different windows correspond to different gel migration experiments.

Figure 4

(A) and (B) show the pattern of ³⁵S-labelled (A) or Coomassie-labelled (B) proteins of nuclear extracts. The spots of interest are indicated by arrows. (C) and (E) are South-western blots of 2D-gels performed with intramolecular 55mer triplexes. The third strand in the polypyrimidine motif, is composed of CBrdU (C, T2), or CT [E, T1(np)]. The control corresponding to annealing of the third strand alone are shown in (D) (S2). Western-blot performed on the same membrane in the presence of specific antibodies raised against hnRNP K, hnRNP A2, and p54nrb (F). The spots of interest are indicated by arrows.