MCM ring hexamerization is a prerequisite for DNA-binding.
MCM ring hexamerization is a prerequisite for DNA-binding.
复制标题
DOI：
10.1093/nar/gkv914
复制
发表时间：
2015-10-30
影响因子：
14.9
通讯作者：
Enemark EJ
中科院分区：
文献类型：
--
作者：
Froelich CA;Nourse A;Enemark EJ
六聚体微型染色体维持 (MCM) 蛋白复合物形成一个环，在真核生物和古细菌的复制叉处解开 DNA。我们最近与单链DNA（ssDNA）结合的古菌MCM N端结构域的晶体结构揭示了ssDNA在紧密的亚基界面上缔合，而不是在松散的界面上缔合，这表明DNA结合不仅受到亚基的DNA结合残基（MCM ssDNA结合基序，MSSB）的控制，而且还受到亚基的相对方向的控制。现在，我们扩展了这些发现，表明古菌强烈火球菌的 MCM N 端结构域与 DNA 的结合特异地以六聚寡聚形式发生。我们表明，六聚化缺陷的突变体在结合 ssDNA 方面存在缺陷，尽管保留了晶体结构中观察到的与 ssDNA 相互作用的所有残基。一种表现出严重缺陷的六聚化和 ssDNA 结合的突变位于保守的苯丙氨酸，该突变与与染色体不稳定、癌症和亚基间关联减少相关的小鼠 Mcm4(Chaos3) 突变一致。
The hexameric Minichromosome Maintenance (MCM) protein complex forms a ring that unwinds DNA at the replication fork in eukaryotes and archaea. Our recent crystal structure of an archaeal MCM N-terminal domain bound to single-stranded DNA (ssDNA) revealed ssDNA associating across tight subunit interfaces but not at the loose interfaces, indicating that DNA-binding is governed not only by the DNA-binding residues of the subunits (MCM ssDNA-binding motif, MSSB) but also by the relative orientation of the subunits. We now extend these findings by showing that DNA-binding by the MCM N-terminal domain of the archaeal organism Pyrococcus furiosus occurs specifically in the hexameric oligomeric form. We show that mutants defective for hexamerization are defective in binding ssDNA despite retaining all the residues observed to interact with ssDNA in the crystal structure. One mutation that exhibits severely defective hexamerization and ssDNA-binding is at a conserved phenylalanine that aligns with the mouse Mcm4(Chaos3) mutation associated with chromosomal instability, cancer, and decreased intersubunit association.