Amino acids of the sulfolobus solfataricus MCM 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 (MthMCM) 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 Mth and Sso MCM sequences identified positively charged amino acids in SsoMCM that could point to the center of the channel. These residues (K129, K134, H146 and K194) 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 SsoMCM are unable to bind DNA either in single- or double-stranded form in band-shift assays and do not display helicase activity. In contrast, 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.