Modular organization of the Sulfolobus solfataricus mini-chromosome maintenance protein

Biagio Pucci, Mariarita De Felice, Monia Rocco, Francesco Esposito, Mariarosaria De Falco, Luca Esposito, Mosé Rossi, Francesca M. Pisani

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Abstract

Mini-chromosome maintenance (MCM) proteins form ring-like hexameric complexes that are commonly believed to act as the replicative DNA helicase at the eukaryotic/archaeal DNA replication fork. Because of their simplified composition with respect to the eukaryotic counterparts, the archaeal MCM complexes represent a good model system to use in analyzing the structural/functional relationships of these important replication factors. In this study the domain organization of the MCM-like protein from Sulfolobus solfataricus (Sso MCM) has been dissected by trypsin partial proteolysis. Three truncated derivatives of Sso MCM corresponding to protease-resistant domains were produced as soluble recombinant proteins and purified: the N-terminal domain (N-ter, residues 1-268); a fragment comprising the AAA+ and C-terminal domains (AAA+-C-ter, residues 269-686); and the C-terminal domain (C-ter, residues 504-686). All of the purified recombinant proteins behaved as monomers in solution as determined by analytical gel filtration chromatography, suggesting that the polypeptide chain integrity is required for stable oligomerization of Sso MCM. However, the AAA+-C-ter derivative, which includes the AAA+ motor domain and retains ATPase activity, was able to form dimers in solution when ATP was present, as analyzed by size exclusion chromatography and glycerol gradient sedimentation analyses. Interestingly, the AAA+-C-ter protein could displace oligonucleotides annealed to M13 single-stranded DNA although with a reduced efficiency in comparison with the full-sized Sso MCM. The implications of these findings for understanding the DNA helicase mechanism of the MCM complex are discussed.

Original languageEnglish
Pages (from-to)12574-12582
Number of pages9
JournalJournal of Biological Chemistry
Volume282
Issue number17
DOIs
Publication statusPublished - Apr 27 2007

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ASJC Scopus subject areas

  • Biochemistry

Cite this

Pucci, B., De Felice, M., Rocco, M., Esposito, F., De Falco, M., Esposito, L., Rossi, M., & Pisani, F. M. (2007). Modular organization of the Sulfolobus solfataricus mini-chromosome maintenance protein. Journal of Biological Chemistry, 282(17), 12574-12582. https://doi.org/10.1074/jbc.M610953200