Beijing National Laboratory for Condensed Matter Physics and CAS Key Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China.
Nucleic Acids Res. 2012 Oct;40(19):9802-14. doi: 10.1093/nar/gks728. Epub 2012 Aug 9.
Bloom (BLM) syndrome is an autosomal recessive disorder characterized by an increased risk for many types of cancers. Previous studies have shown that BLM protein forms a hexameric ring structure, but its oligomeric form in DNA unwinding is still not well clarified. In this work, we have used dynamic light scattering and various stopped-flow assays to study the active form and kinetic mechanism of BLM in DNA unwinding. It was found that BLM multimers were dissociated upon ATP hydrolysis. Steady-state and single-turnover kinetic studies revealed that BLM helicase always unwound duplex DNA in the monomeric form under conditions of varying enzyme and ATP concentrations as well as 3'-ssDNA tail lengths, with no sign of oligomerization being discerned. Measurements of ATPase activity further indicated that BLM helicase might still function as monomers in resolving highly structured DNAs such as Holliday junctions and D-loops. These results shed new light on the underlying mechanism of BLM-mediated DNA unwinding and on the molecular and functional basis for the phenotype of heterozygous carriers of BLM syndrome.
布卢姆综合征（BLM）是一种常染色体隐性遗传病，其特征是多种癌症的风险增加。先前的研究表明，BLM 蛋白形成六聚体环结构，但在 DNA 解旋过程中的寡聚形式仍未得到很好的阐明。在这项工作中，我们使用动态光散射和各种停流测定法研究了 BLM 在 DNA 解旋中的活性形式和动力学机制。结果发现，BLM 多聚体在 ATP 水解时解离。稳态和单轮动力学研究表明，BLM 解旋酶在不同酶和 ATP 浓度以及 3'-ssDNA 尾长条件下，总是以单体形式解旋双链 DNA，没有发现寡聚化的迹象。ATPase 活性的测量进一步表明，BLM 解旋酶在解析高度结构化的 DNA （如 Holliday 连接和 D 环）时，可能仍然以单体形式发挥作用。这些结果为 BLM 介导的 DNA 解旋的潜在机制以及 BLM 综合征杂合子携带者表型的分子和功能基础提供了新的认识。
