Analytical biochemistry of DNA--protein assemblies from crude cell extracts.
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.
Hégarat
Nadia
N
INSERM, U565, USM503, Département de Régulations, développement et diversité moléculaire, Laboratoire des Régulations et dynamique des génomes, CNRS, UMR5153, Acides nucléiques: dynamique, ciblage et fonctions biologiques, Paris, France.
Cardoso
Gildas Mouta
GM
Rusconi
Filippo
F
Francois
Jean-Christophe
JC
Praseuth
Danièle
D
D002457:Cell Extracts / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D045643:DNA Repair Enzymes / Q000302:isolation & purification / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D009841:Oligonucleotides / Q000737:chemistry; D010777:Photochemistry; D012097:Repressor Proteins / Q000302:isolation & purification
D002457:Cell Extracts; D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D045643:DNA Repair Enzymes