Characterization of a Complex Involved In Rothmund-Thomson Syndrome

Rothmund Thomson综合征(RTS)是一种罕见的疾病，与基因组不稳定、癌症易感性、皮肤和骨骼异常以及过早衰老的某些特征有关。这种疾病是由RECQL4突变引起的，RECQL4是RecQ家族的一种假定解旋酶。为了了解这种疾病的发病机制，我们纯化了一个人RECQL4复合体，并鉴定了它的相关成分。RECQL4主要存在于HeLa细胞的细胞质提取物中，而不是主要有核的BLM和WRN解旋酶。然而，在未转化的Wi-38成纤维细胞中，RECQL4被发现主要是有核的，并且在转化的HeLa细胞中的总水平显著低于转化的HeLa细胞。从HeLa细胞中分离到RECQL4，它与UBR1和UBR2形成稳定的复合体。这些200 kD的蛋白质是N末端规则通路的泛素连接酶，其底物包括促进蛋白质转换的蛋白质。这种蛋白分解途径的功能包括调节多肽输入、染色体稳定性、减数分裂、细胞凋亡和心血管发育。虽然UBR1和UBR2的已知作用是介导其底物的多泛素化(和随后的降解)，但UBR1/2结合的RECQL4在体内不是泛素化的，在HeLa细胞中是一个长期存在的蛋白质。我们的数据与N端规则途径的RECQL4和泛素连接酶一致，有助于维持基因组稳定。 我们还纯化了含有RECQL5的复合体，RECQL5是另一种参与维持基因组稳定的RecQ解旋酶。线虫中RecQL5基因的缺失使寿命减少了37%，而在小鼠中该基因的失活会导致基因组不稳定和癌症风险增加。为了阐明其作用机制，我们纯化了一个与RecQL5相关的复合体，并鉴定其主要成分为RNA聚合酶II(POL II)。生物信息学和结构建模指导的突变发现，RecQL5中有两个保守的区域，即KIX和SRI结构域，它们已经被认为是POL II的转录调节因子。RecQL5-KIX结构域结合聚合酶的起始形式(POL IIa)和延伸形式(POL IIO)，而RecQL5-SRI结构域只与延伸形式相互作用。完全功能的RecQL5必须同时具有解旋酶活性和与起始聚合酶的结合，因为缺乏这两种活性的突变株在抑制姐妹染色单体交换和抵抗喜树碱诱导的DNA损伤方面存在部分缺陷，而缺乏这两种活性的突变株是完全缺陷的。我们的数据表明，RecQL5通过两种平行的机制促进基因组的稳定：作为一种RecQ解旋酶参与依赖于同源重组的DNA修复，以及通过调节POL II的启动来减少转录相关的复制损伤和重组。 我们发现RecQL5还与其他几个对DNA复制和修复至关重要的分子相互作用。其中包括RAD51重组酶和pncA(DNA聚合酶的夹子)。我们已经将它们的相互作用结构域映射到RecQL5中，并表明这些相互作用失活的突变体降低了抑制姐妹染色单体交换和抵抗Campothecin介导的细胞杀伤的能力。这些研究表明，RecQL5通过多种机制参与保护基因组的完整性。 我们与Patrick Sungs小组合作，专门表征了RecQL5的RAD51-相互作用结构域。我们发现，RecQL5在体外可以调节RAD51重组酶的活性，而失去与RAD51相互作用能力的RecQL5点突变株不能调节其重组酶的活性。相同的点突变也不能抑制非法重组，也不能抵抗喜树碱诱导的细胞杀伤。我们的数据表明，RecQL5的一个机制是调节RAD51介导的同源重组依赖的DNA修复。 在一项DNA损伤起源的研究中，我们参与了一个与H·程博士合作的项目，研究线粒体如何产生可以修改DNA的活性氧。 我们与其他同事合作，研究了五种人类RecQ螺旋酶与三个特征良好的人类复制起源在体内的关联。我们发现，在未受干扰的细胞中，只有RECQ1(也称为RECQL或RECQL1)和RECQ4(也称为RECQL4)以细胞周期调节的方式与复制起点相关。在ORC和MCM复合体组装后，RECQL4在G(1)末期被招募到原点，而RECQ1和额外的RECQL4在S阶段开始时在原点被加载，此时许可的原点开始激发。在DNA复制开始后，这两种蛋白质都从起源上丢失，表明要么解体，要么跟踪新形成的复制体。RECQ1耗尽后，新生起源DNA合成和起始激发频率降低，RECQL4耗尽后下降程度更大。RECQ1的缺失，虽然不是RECQL4的缺失，但也抑制了原本未受干扰的细胞中的复制分叉率。这些结果表明，RECQ1和RECQL4与人类DNA复制复合体结合，在体内DNA复制起始和复制分叉过程中具有独特的作用。

Rothmund Thomson Syndromes (RTS) is a rare disease associated with genome instability, predisposition to cancer, skin and skeletal abnormalities, and some features of premature aging. The disease is caused by mutation in RECQL4, a putative helicase of the RecQ family. To understand the mechanism of this disease, we have purified a human RECQL4 complex and identified its associated components. The bulk of RECQL4 was present in cytoplasmic extracts of HeLa cells, in contrast to the largely nuclear BLM and WRN helicases. However, in untransformed WI-38 fibroblasts, RECQL4 was found to be largely nuclear, and was present at significantly lower total levels than in transformed HeLa cells. RECQL4 from HeLa cells was isolated as a stable complex with UBR1 and UBR2. These 200 kD proteins are ubiquitin ligases of the N-end rule pathway, whose substrates include proteins that promote protein turnovers. The functions of this proteolytic pathway include the regulation of peptide import, chromosome stability, meiosis, apoptosis, and cardiovascular development. Although the known role of UBR1 and UBR2 is to mediate polyubiquitylation (and subsequent degradation) of their substrates, the UBR1/2-bound RECQL4 was not ubiquitylated in vivo, and was a long-lived protein in HeLa cells. Our data are consistent with RECQL4 and ubiquitin ligases of the N-end rule pathway contributing to maintaining genomic stability. We have also purified complexes containing RECQL5, another RecQ helicase involved in maintaining genome stability. Deficiency of RecQL5 in C.elegans reduces life-span by 37%, and inactivation of this gene in mice results genomic instability and increased cancer risks. To elucidate its mechanism of action, we purified a RecQL5-associated complex and identified its major component as RNA polymerase II (Pol II). Bioinformatics and structural modeling-guided mutagenesis revealed two conserved regions in RecQL5 as KIX and SRI domains, already known as transcriptional regulators for Pol II. The RecQL5-KIX domain binds both initiation (Pol IIa) and elongation (Pol IIo) forms of the polymerase, whereas the RecQL5-SRI domain interacts only with the elongation form. Fully functional RecQL5 must have both helicase activity and association with the initiation polymerase, because mutants lacking either activity are partially defective in the suppression of sister chromatid exchange and resistance to camptothecin-induced DNA damage, and mutants lacking both activities are completely defective. Our data suggest that RecQL5 promotes genome stabilization through two parallel mechanisms: by participation in homologous recombination-dependent DNA repair as a RecQ helicase and by regulating the initiation of Pol II to reduce transcription-associated replication impairment and recombination. We found that RecQL5 also interacts with several other molecules important for DNA replication and repair. These include the RAD51 recombinase and PNCA (a clamp for DNA polymerase). We have mapped their interaction domains within RecQL5, and shown that mutants that are inactivated of these interactions have reduced capacity in suppressing SCE and resist campothecin-mediated cell killing. These studies suggest that RecQL5 participates in protecting genome integrity through multiple mechanisms. We specifically characterized the RAD51-interaction domain of RecQL5 in collaboration with Patrick Sungs group. We found that RecQL5 can regulate RAD51 recombinase activity in vitro, whereas RecQL5 point mutants that lost the ability to interact with RAD51 failed to regulate its recombinase activity. The same point mutants also failed to suppress illegitimate recombination and resist campothecin-induced cell killing. Our data suggest that one mechanism of RecQL5 is to regulate homologous recombination dependent DNA repair mediated by RAD51. In a study of the origin of DNA damage, we participated in a collaborative project with Dr. H. Chengs group to study how mitochondria produce reactive oxygen that can modify DNA. We collaborated with other colleagues to investigate the in vivo association of the five human RecQ helicases with three well-characterized human replication origins. We show that only RECQ1 (also called RECQL or RECQL1) and RECQ4 (also called RECQL4) associate with replication origins in a cell cycle-regulated fashion in unperturbed cells. RECQL4 is recruited to origins at late G(1), after ORC and MCM complex assembly, while RECQ1 and additional RECQL4 are loaded at origins at the onset of S phase, when licensed origins begin firing. Both proteins are lost from origins after DNA replication initiation, indicating either disassembly or tracking with the newly formed replisome. Nascent-origin DNA synthesis and the frequency of origin firing are reduced after RECQ1 depletion and to a greater extent after RECQL4 depletion. Depletion of RECQ1, though not that of RECQL4, also suppresses replication fork rates in otherwise unperturbed cells. These results indicate that RECQ1 and RECQL4 associate with the human DNA replication complex and have distinctive roles in DNA replication initiation and replication fork progression in vivo.