RESUMEN
Mcm proteins play an essential role in eukaryotic DNA replication, but their biochemical functions are poorly understood. Recently, we reported that a DNA helicase activity is associated with an Mcm4-Mcm6-Mcm7 (Mcm4,6,7) complex, suggesting that this complex is involved in the initiation of DNA replication as a DNA-unwinding enzyme. In this study, we have expressed and isolated the mouse Mcm2, 4,6,7 proteins from insect cells and characterized various mutant Mcm4,6,7 complexes in which the conserved ATPase motifs of the Mcm4 and Mcm6 proteins were mutated. The activities associated with such preparations demonstrated that the DNA helicase activity is intrinsically associated with the Mcm4,6,7 complex. Biochemical analyses of these mutant Mcm4,6,7 complexes indicated that the ATP binding activity of the Mcm6 protein in the complex is critical for DNA helicase activity and that the Mcm4 protein may play a role in the single-stranded DNA binding activity of the complex. The results also indicated that the two activities of DNA helicase and single-stranded DNA binding can be separated.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfatasas/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/aislamiento & purificación , Proteínas Cromosómicas no Histona , ADN Helicasas/genética , ADN Helicasas/aislamiento & purificación , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Expresión Génica , Ratones , Componente 2 del Complejo de Mantenimiento de Minicromosoma , Componente 4 del Complejo de Mantenimiento de Minicromosoma , Componente 6 del Complejo de Mantenimiento de Minicromosoma , Componente 7 del Complejo de Mantenimiento de Minicromosoma , Mutagénesis , Proteínas Nucleares/genética , Proteínas Nucleares/aislamiento & purificación , Estructura Terciaria de ProteínaRESUMEN
Minichromosome maintenance (MCM) proteins play an essential role in eukaryotic DNA replication and bind to chromatin before the initiation of DNA replication. We reported that MCM protein complexes consisting of MCM2, -4, -6, and -7 bind strongly to a histone-Sepharose column (Ishimi, Y., Ichinose, S., Omori, A., Sato, K., and Kimura, H. (1996) J. Biol. Chem. 271, 24115-24122). Here, we have analyzed this interaction at the molecular level. We found that among six mouse MCM proteins, only MCM2 binds to histone; amino acid residues 63-153 are responsible for this binding. The region required for nuclear localization of MCM2 was mapped near this histone-binding domain. Far-Western blotting analysis of truncated forms of H3 histone indicated that amino acid residues 26-67 of H3 histone are required for binding to MCM2. We have also shown that mouse MCM2 can inhibit the DNA helicase activity of the human MCM4, -6, and -7 protein complex. These results suggest that MCM2 plays a different role in the initiation of DNA replication than the other MCM proteins.