Differences in the single-stranded DNA binding activities of MCM2-7 and MCM467: MCM2 and MCM5 define a slow ATP-dependent step.
The MCM2-7 complex, a hexamer containing six distinct and essential subunits, is postulated to be the eukaryotic replicative DNA helicase. Although all six subunits function at the replication fork, only a specific subcomplex consisting of the MCM4, 6, and 7 subunits (MCM467) and not the MCM2-7 complex exhibits DNA helicase activity in vitro. To understand why MCM2-7 lacks helicase activity and to address the possible function of the MCM2, 3, and 5 subunits, we have compared the biochemical properties of the Saccharomyces cerevisiae MCM2-7 and MCM467 complexes. We demonstrate that both complexes are toroidal and possess a similar ATP-dependent single-stranded DNA (ssDNA) binding activity, indicating that the lack of helicase activity by MCM2-7 is not due to ineffective ssDNA binding. We identify two important differences between them. MCM467 binds dsDNA better than MCM2-7. In addition, we find that the rate of MCM2-7/ssDNA association is slow compared with MCM467; the association rate can be dramatically increased either by preincubation with ATP or by inclusion of mutations that ablate the MCM2/5 active site. We propose that the DNA binding differences between MCM2-7 and MCM467 correspond to a conformational change at the MCM2/5 active site with putative regulatory significance.
Bochman
Matthew L
ML
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260.
Schwacha
Anthony
A
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260. Electronic address: schwacha@pitt.edu.
D000255:Adenosine Triphosphate / Q000737:chemistry*; D001665:Binding Sites; D001667:Binding, Competitive; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000502:physiology*; D002868:Chromosomal Proteins, Non-Histone; D004265:DNA Helicases / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D005656:Fungal Proteins / Q000502:physiology*; D007700:Kinetics; D064148:Minichromosome Maintenance Complex Component 4; D064151:Minichromosome Maintenance Complex Component 6; D008954:Models, Biological; D008968:Molecular Conformation; D009711:Nucleotides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000502:physiology*; D013997:Time Factors
D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C069200:MCM5 protein, S cerevisiae; D009711:Nucleotides; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D004265:DNA Helicases; C069199:MCM2 protein, S cerevisiae; C092802:MCM4 protein, S cerevisiae; C491207:MCM6 protein, S cerevisiae; D064148:Minichromosome Maintenance Complex Component 4; D064151:Minichromosome Maintenance Complex Component 6