Analytical biochemistry of DNA--protein assemblies from crude cell extracts

Abstract

Purification of specific DNA-protein complexes is a challenging task, as the involved interactions can be both electrostatic/H-bond and hydrophobic. The chromatographic stringency needed to obtain reasonable purifications uses salts and detergents. However, these components elicit the removal of proteins unspecifically bound to the chromatographic support itself, thus contaminating the purification products. In this work, a photocleavable linker connected the target oligonucleotidic sequence to the chromatographic beads so as to allow the irradiation-based release of the purified DNA-protein complexes off the beads. Our bioanalytical conditions were validated by purifying the tetracycline repressor protein onto a specific oligonucleotide. The purification factor was unprecedented, with a single contaminant. The robustness of our method was challenged by applying it to the purification of multiprotein assemblies forming onto DNA damage-mimicking oligonucleotides. The purified components were identified as well-known DNA repair proteins, and were shown to retain their enzymatic activities, as seen by monitoring DNA ligation products. Remarkably, kinase activities, also monitored, were found to be distinct on the beads and on the purified DNA-protein complexes, showing the benefits to uncouple the DNA-protein assemblies from the beads for a proper understanding of biochemical regulatory mechanisms involved in the DNA-protein assemblies.

Figure 1.

Overview of the nucleic acid-binding protein purification strategy. (A) Schematic representation of the chemistry involved in the preparation of the chromatographic resin. The 5′P of the oligonucleotide is conjugated to the photo-reactive group coupled itself to a biotin through a C₆ spacer. The oligonucleotide is incubated with streptadivin-coated magnetic beads to yield the chromatographic solid phase. (B) Purification procedure. Protein extracts are incubated with the chromatographic resin prepared in (A). After magnetic harvesting of the beads, washes are performed. Photocleavage of the linker, with near UV-light irradiation, releases the DNA–protein complexes, thus minimizing their contamination by the proteins unspecifically bound to the beads.

Figure 2.

EMSA analysis of fractions obtained from nucleic acid-binding protein purification. (A) To observe the band shift corresponding to the TetR-TetO interaction, whole cell extracts were mixed with the PCB-TetO bait in absence or in presence of tetracycline (Tet; lanes 2 and 3, respectively). To perform the purification, whole cell extract was incubated with the PCB-TetO immobilized on beads, then the presence of the complex was analyzed in the purification product (lane 4). (B) Tetracycline was mixed with the purification product to confirm the specificity of the DNA–protein interaction. The asterisk indicates that only 1/2 of the corresponding fraction was loaded.

Figure 3.

Purification of the tetracycline repressor protein from whole human cell extracts by affinity for its cognate sequence. Whole cell extracts were mixed with the chromatographic phase followed by washes. (A) Supernatant and pellet fractions obtained from irradiated and non-irradiated samples were analyzed by SDS-PAGE gel electrophoresis and proteins were blotted onto a membrane and revealed by Sypro blot-staining (left panel) and subsequently anti-TetR antibody-staining (right panel). (B) 2D-gel electrophoresis of proteins released after detergent treatment of the whole chromatographic material (left panel) and of DNA–protein complexes recovered in the supernatant after irradiation (right panel). The proteins were stained with Sypro Ruby. The arrows point to the TetR spot. The asterisk indicate that only 1/3 of the pellet fractions was loaded. MW, molecular weight markers.

Figure 4.

Affinity purification of the DNA ends-binding proteins. The proteins were purified from HeLa nuclear extracts using the PCB-TetO target sequence immobilized on beads (lanes 2 and 4) and without oligonucleotide on beads as control (lane 3). (A) The chromatographic slurry was either treated with detergents (lane 2) or irradiated (lanes 3 and 4), then the released proteins were separated on an 8% SDS-PAGE gel and stained with Sypro Ruby protein gel stain. (B) The purification product obtained after irradiation was analyzed by western blot with antibodies against the DNA ends-binding proteins as indicated. MW, molecular weight markers.

Figure 5.

Oligonucleotide ligation by DNA ends-binding protein complexes. After washes, the DNA–protein assemblies were incubated with ATP to allow the ligation of the TetO sequence. In lane 3, the ligation was performed on the beads in presence of ATP then the nucleic sequence was released after irradiation of the chromatographic slurry. In lane 4, the DNA–protein assemblies were recovered after the irradiation step then incubated with ATP. The oligonucleotides were resolved on an 8% non-denaturing polyacrylamide gel and revealed by Southern blot with the radiolabeled non-photocleavable strand of the PCB-TetO duplex. Lane 1 shows the intact TetO oligonucleotide and lane 2 the oligonucleotide ligated by the T4 DNA ligase protein.

Figure 6.

Analysis of the phosphorylation of the XRCC4 protein. (A) HeLa nuclear extracts were depleted in ATP with hexokinase and glucose then incubated with the oligonucleotide immobilized on beads. After washes, the chromatographic slurry was incubated with ATP for 2 h at 30°C then irradiated (lanes 4 and 6) or first irradiated then incubated with ATP (lanes 5 and 7). To determine the involved kinases, the proteins were pre-incubated with 10 μM of wortmannin (lanes 6 and 7) right before the incubation with ATP. Controls were performed in absence of ATP (lanes 2 and 3). The proteins from HeLa nuclear extracts depleted in ATP and from the purification products were resolved on an 8% SDS-PAGE gel then transferred on a nitrocellulose membrane and immunoblotted with a polyclonal rabbit anti-XRCC4 antibody. (B) HeLa nuclear extract depleted in ATP (lanes 1, 8 and 9) and the purification product (lanes 2–7, 10 and 11) incubated with ATP were treated with calf intestine phosphatase (CIP) to detect the phosphorylated forms of the XRCC4 protein. An asterisk indicates a possible proteolytic product of the XRCC4 protein. Symbols (filled and empty circles, filled square) indicate XRCC4 phosphorylated variants.