Kinoshita Yayoi, Johnson Edward M, Gordon Ronald E, Negri-Bell Heather, Evans Mark T, Coolbaugh Jennifer, Rosario-Peralta Yaseris, Samet Joseph, Slusser Evelyn, Birkenbach Mark P, Daniel Dianne C
Department of Pathology, Mount Sinai School of Medicine, New York, New York 10029, USA.
Microsc Res Tech. 2008 Apr;71(4):288-97. doi: 10.1002/jemt.20553.
Minichromosome maintenance (MCM) proteins are essential for DNA replication in eukaryotes. A subcomplex of the MCM2-7 family members, initially characterized in yeast, is thought to serve as a eukaryotic DNA replicative helicase. MCM8 is a new family member, not present in yeast, which may function alone or with other family members in aspects of DNA metabolism, including replication initiation and elongation. Through the use of chromatin immunoprecipitation, we find that MCM8, like MCM7, colocalizes on a specific DNA segment of the c-MYC replication initiation zone (c-MYC replicator) with Cdc6, a protein potentially involved in loading MCM proteins onto DNA. The association between MCM8 and MCM7 peaks in mid G1, at the time of assembly of the prereplication complex. The association of both MCM proteins with Cdc6, however, continues even after DNA replication is complete. We also find that MCM8 colocalizes at the c-MYC replicator with chromatin-bound Cdk2. Our data indicate that any role MCM8 may play in elongation is likely to be discontinuous, in its association with DNA, from a potential role in initiation. Using immunogold electron microscopy we show that MCM8 and MCM7 differ in spatial relation to RPA70 during S phase. Our data strongly suggest that MCM8 functions with other known replication proteins in processes which accompany DNA replication, especially initiation, and which are specifically adapted to suit higher eukaryotes.
微小染色体维持（MCM）蛋白对于真核生物中的DNA复制至关重要。MCM2 - 7家族成员的一个亚复合物最初在酵母中被鉴定，被认为是真核生物的DNA复制解旋酶。MCM8是一个新的家族成员，不存在于酵母中，它可能在DNA代谢的各个方面单独发挥作用或与其他家族成员共同发挥作用，包括复制起始和延伸。通过染色质免疫沉淀技术，我们发现MCM8与MCM7一样，与Cdc6共定位于c - MYC复制起始区（c - MYC复制子）的特定DNA片段上，Cdc6是一种可能参与将MCM蛋白加载到DNA上的蛋白质。MCM8与MCM7之间的关联在G1中期达到峰值，此时是前复制复合物组装的时候。然而，即使在DNA复制完成后，这两种MCM蛋白与Cdc6的关联仍在继续。我们还发现MCM8与染色质结合的Cdk2在c - MYC复制子处共定位。我们的数据表明，MCM8在延伸过程中可能发挥的任何作用，在其与DNA的关联方面，可能与起始过程中的潜在作用不连续。通过免疫金电子显微镜我们显示，在S期MCM8和MCM7与RPA70的空间关系不同。我们的数据强烈表明，MCM8在伴随DNA复制的过程中，特别是在起始过程中，与其他已知的复制蛋白一起发挥作用，并且这些过程是专门为适应高等真核生物而进行的。
