Chromatographic isolation of the functionally active MutS protein covalently linked to deoxyribonucleic acid.
DNA metabolism is based on formation of different DNA-protein complexes which can adopt various conformations. To characterize functioning of such complexes, one needs a solution-based technique which allows fixing a complex in a certain transient conformation. The crosslinking approach is a popular tool for such studies. However, it is under debate if the protein components retain their natural activities in the resulting crosslinked complexes. In the present work we demonstrate the possibility of obtaining pure DNA conjugate with functionally active protein using as example MutS protein from Escherichia coli mismatch repair system. A conjugate of a chemically modified mismatch-containing DNA duplex with MutS is fixed by thiol-disulfide exchange reaction. To perform a reliable test of the protein activity in the conjugate, such conjugate must be thoroughly separated from the uncrosslinked protein and DNA prior to the test. In the present work, we employ anion exchange chromatography for this purpose for the first time and demonstrate this technique to be optimal for the conjugate purification. The activity test is a FRET-based detection of DNA unbending. We show experimentally that MutS in the conjugate retains its ability to unbend DNA in response to ATP addition and find out for the first time that the DNA unbending rate increases with increasing ATP concentration. Since the crosslinked complexes contain active MutS protein, they can be used in further experiments to investigate MutS interactions with other proteins of the mismatch repair system.
Chemistry Department and A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskie Gory 1, 119991 Moscow, Russia.
Mayya
M
Monakhova
Chemistry Department and A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskie Gory 1, 119991 Moscow, Russia.
Alexandra
A
Ryazanova
Institute for Biochemistry, FB 08, Justus Liebig University, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany.
Andreas
A
Hentschel
Chemistry Department and A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskie Gory 1, 119991 Moscow, Russia. Electronic address: viryasovmb@gmail.com.
Mikhail
M
Viryasov
Chemistry Department and A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskie Gory 1, 119991 Moscow, Russia.
Tatiana
T
Oretskaya
Institute for Biochemistry, FB 08, Justus Liebig University, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany.
Peter
P
Friedhoff
Chemistry Department and A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskie Gory 1, 119991 Moscow, Russia.
Elena
E
Kubareva
D000255:Adenosine Triphosphate / Q000378:metabolism; D001671:Biochemistry / Q000379:methods*; D004247:DNA / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D051719:MutS DNA Mismatch-Binding Protein / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding
D000255:Adenosine Triphosphate; D004247:DNA; D051719:MutS DNA Mismatch-Binding Protein
Anion exchange chromatography; DNA–protein crosslinking; Mismatch repair; MutS protein