Amino acids of the Sulfolobus solfataricus mini-chromosome maintenance-like DNA helicase involved in DNA binding/remodeling.
Herein we report the identification of amino acids of the Sulfolobus solfataricus mini-chromosome maintenance (MCM)-like DNA helicase (SsoMCM), which are critical for DNA binding/remodeling. The crystallographic structure of the N-terminal portion (residues 2-286) of the Methanothermobacter thermoautotrophicum MCM protein revealed a dodecameric assembly with two hexameric rings in a head-to-head configuration and a positively charged central channel proposed to encircle DNA molecules. A structure-guided alignment of the M. thermoautotrophicum and S. solfataricus MCM sequences identified positively charged amino acids in SsoMCM that could point to the center of the channel. These residues (Lys-129, Lys-134, His-146, and Lys-194) were changed to alanine. The purified mutant proteins were all found to form homo-hexamers in solution and to retain full ATPase activity. K129A, H146A, and K194A SsoMCMs are unable to bind DNA either in single- or double-stranded form in band shift assays and do not display helicase activity. In contrast, the substitution of lysine 134 to alanine affects only binding to duplex DNA molecules, whereas it has no effect on binding to single-stranded DNA and on the DNA unwinding activity. These results have important implications for the understanding of the molecular mechanism of the MCM DNA helicase action.
Pucci
Biagio
B
Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131 Napoli, Italy.
De Felice
Mariarita
M
Rossi
Mosè
M
Onesti
Silvia
S
Pisani
Francesca M
FM
D000251:Adenosine Triphosphatases / Q000737:chemistry; D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D002845:Chromatography; D002850:Chromatography, Gel; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry*; D004265:DNA Helicases / Q000737:chemistry*; D019992:Databases as Topic; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D017013:Methanobacteriaceae / Q000378:metabolism; D026341:Minichromosome Maintenance 1 Protein / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D048229:Sulfolobus solfataricus / Q000378:metabolism*
D000596:Amino Acids; D026341:Minichromosome Maintenance 1 Protein; D009841:Oligonucleotides; D011994:Recombinant Proteins; D006639:Histidine; D004247:DNA; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D008239:Lysine; D000409:Alanine