Physical and functional interaction between the mini-chromosome maintenance-like DNA helicase and the single-stranded DNA binding protein from the crenarchaeon Sulfolobus solfataricus.
Mini-chromosome Maintenance (MCM) proteins play an essential role in both initiation and elongation phases of DNA replication in Eukarya. Genes encoding MCM homologs are present also in the genomic sequence of Archaea and the MCM-like protein from the euryarchaeon Methanobacterium thermoautotrophicum (Mth MCM) was shown to possess a robust ATP-dependent 3'-5' DNA helicase activity in vitro. Herein, we report the first biochemical characterization of a MCM homolog from a crenarchaeon, the thermoacidophile Sulfolobus solfataricus (Sso MCM). Gel filtration and glycerol gradient centrifugation experiments indicate that the Sso MCM forms single hexamers (470 kDa) in solution, whereas the Mth MCM assembles into double hexamers. The Sso MCM has NTPase and DNA helicase activity, which preferentially acts on DNA duplexes containing a 5'-tail and is stimulated by the single-stranded DNA binding protein from S. solfataricus (Sso SSB). In support of this functional interaction, we demonstrated by immunological methods that the Sso MCM and SSB form protein.protein complexes. These findings provide the first in vitro biochemical evidence of a physical/functional interaction between a MCM complex and another replication factor and suggest that the two proteins may function together in vivo in important DNA metabolic pathways.
Carpentieri
Floriana
F
Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Via P. Castellino 111, Napoli 80131, Italy.
De Felice
Mariarita
M
De Falco
Mariarosaria
M
Rossi
Mose
M
Pisani
Francesca M
FM
D000251:Adenosine Triphosphatases / Q000378:metabolism; D015153:Blotting, Western; D002498:Centrifugation; D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D002875:Chromosomes / Q000378:metabolism*; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D026341:Minichromosome Maintenance 1 Protein / Q000378:metabolism*; D009711:Nucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D016944:Sulfolobus / Q000378:metabolism*; D013997:Time Factors
D004268:DNA-Binding Proteins; D026341:Minichromosome Maintenance 1 Protein; D009711:Nucleotides; D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases; D004265:DNA Helicases