Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224, USA.
J Biol Chem. 2012 Jan 6;287(2):1007-21. doi: 10.1074/jbc.M111.276022. Epub 2011 Nov 18.
Mutations in the human ChlR1 gene are associated with a unique genetic disorder known as Warsaw breakage syndrome characterized by cellular defects in sister chromatid cohesion and hypersensitivity to agents that induce replication stress. A role of ChlR1 helicase in sister chromatid cohesion was first evidenced by studies of the yeast homolog Chl1p; however, its cellular functions in DNA metabolism are not well understood. We carefully examined the DNA substrate specificity of purified recombinant human ChlR1 protein and the biochemical effect of a patient-derived mutation, a deletion of a single lysine (K897del) in the extreme C terminus of ChlR1. The K897del clinical mutation abrogated ChlR1 helicase activity on forked duplex or D-loop DNA substrates by perturbing its DNA binding and DNA-dependent ATPase activity. Wild-type ChlR1 required a minimal 5' single-stranded DNA tail of 15 nucleotides to efficiently unwind a simple duplex DNA substrate. The additional presence of a 3' single-stranded DNA tail as short as five nucleotides dramatically increased ChlR1 helicase activity, demonstrating the preference of the enzyme for forked duplex structures. ChlR1 unwound G-quadruplex (G4) DNA with a strong preference for a two-stranded antiparallel G4 (G2') substrate and was only marginally active on a four-stranded parallel G4 structure. The marked difference in ChlR1 helicase activity on the G4 substrates, reflected by increased binding to the G2' substrate, distinguishes ChlR1 from the sequence-related FANCJ helicase mutated in Fanconi anemia. The biochemical results are discussed in light of the known cellular defects associated with ChlR1 deficiency.
人类 ChlR1 基因突变与一种独特的遗传疾病有关，这种疾病被称为华沙断裂综合征，其特征是姐妹染色单体的黏合细胞缺陷和对诱导复制应激的药物高度敏感。酵母同源物 Chl1p 的研究首次证明了 ChlR1 解旋酶在姐妹染色单体黏合中的作用；然而，其在 DNA 代谢中的细胞功能尚不清楚。我们仔细研究了纯化的重组人 ChlR1 蛋白的 DNA 底物特异性，以及一个源自患者的突变（ChlR1 极端 C 末端单个赖氨酸的缺失）的生化效应。K897del 临床突变通过干扰 ChlR1 的 DNA 结合和 DNA 依赖的 ATP 酶活性，破坏了 ChlR1 解旋酶对分叉双链体或 D 环 DNA 底物的活性。野生型 ChlR1 需要一个最小的 5'单链 DNA 尾巴，长度为 15 个核苷酸，才能有效地解开简单的双链体 DNA 底物。额外存在一个短至五个核苷酸的 3'单链 DNA 尾巴会极大地增加 ChlR1 解旋酶的活性，表明该酶对分叉双链体结构的偏好。ChlR1 优先解开双链体的 G-四链体 (G4) DNA，G2' 底物是最优选的双链体反平行 G4 底物，而四链体平行 G4 结构的活性则相对较低。ChlR1 在 G4 底物上的解旋酶活性差异显著，反映在对 G2' 底物的结合增加，这将 ChlR1 与在范可尼贫血中突变的序列相关的 FANCJ 解旋酶区分开来。这些生化结果与 ChlR1 缺乏相关的已知细胞缺陷进行了讨论。
