Amino acids of the Sulfolobus solfataricus mini-chromosome maintenance-like DNA helicase involved in DNA binding/remodeling

Biagio Pucci, Mariarita De Felice, Mosè Rossi, Silvia Onesti, Francesca M. Pisani

Research output: Contribution to journalArticlepeer-review

Abstract

Herein we report the identification of amino acids of the Sulfolobus solfataricus mini-chromosome maintenance (HCM)-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 (Lye-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.

Original languageEnglish
Pages (from-to)49222-49228
Number of pages7
JournalJournal of Biological Chemistry
Volume279
Issue number47
DOIs
Publication statusPublished - Nov 19 2004

ASJC Scopus subject areas

  • Biochemistry

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