title: Mutational analysis of an archaeal minichromosome maintenance protein exterior hairpin reveals critical residues for helicase activity and DNA binding.
abstract: BACKGROUND
  The mini-chromosome maintenance protein (MCM) complex is an essential replicative helicase for DNA replication in Archaea and Eukaryotes. While the eukaryotic complex consists of six homologous proteins (MCM2-7), the archaeon Sulfolobus solfataricus has only one MCM protein (ssoMCM), six subunits of which form a homohexamer. We have recently reported a 4.35A crystal structure of the near full-length ssoMCM. The structure reveals a total of four beta-hairpins per subunit, three of which are located within the main channel or side channels of the ssoMCM hexamer model generated based on the symmetry of the N-terminal Methanothermobacter thermautotrophicus (mtMCM) structure. The fourth beta-hairpin, however, is located on the exterior of the hexamer, near the exit of the putative side channels and next to the ATP binding pocket.
  
  
  RESULTS
  In order to better understand this hairpin's role in DNA binding and helicase activity, we performed a detailed mutational and biochemical analysis of nine residues on this exterior beta-hairpin (EXT-hp). We examined the activities of the mutants related to their helicase function, including hexamerization, ATPase, DNA binding and helicase activities. The assays showed that some of the residues on this EXT-hp play a role for DNA binding as well as for helicase activity.
  
  
  CONCLUSIONS
  These results implicate several current theories regarding helicase activity by this critical hexameric enzyme. As the data suggest that EXT-hp is involved in DNA binding, the results reported here imply that the EXT-hp located near the exterior exit of the side channels may play a role in contacting DNA substrate in a manner that affects DNA unwinding.
authors: Molecular and Computational Biology, University of Southern California, Los Angeles, CA 90089, USA.
authors: Aaron S
authors: AS
authors: Brewster
authors: Ian M
authors: IM
authors: Slaymaker
authors: Samir A
authors: SA
authors: Afif
authors: Xiaojiang S
authors: XS
authors: Chen
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004252:DNA Mutational Analysis; D004261:DNA Replication; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary / Q000235:genetics*; D016415:Sequence Alignment; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics
chemical_list: D019843:Archaeal Proteins; D004247:DNA; D000251:Adenosine Triphosphatases; D004265:DNA Helicases