METNASE ROLES IN NHEJ, DNA INTEGRATION AND TRANSLOCATION

METNASE 在 NHEJ、DNA 整合和易位中的作用

• 批准号: 7760561
• 负责人: Jac A Nickoloff
• 金额: $ 29.11万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7760561
• 关键词: Binding; Biological Assay; Cancer Etiology; Cell physiology; Cells; Cellular Stress Response; Chemotherapy-Oncologic Procedure; Chromosomal translocation; Chromosomes; DNA; DNA Damage; DNA Double Strand Break; DNA Integration; DNA ligase IV; DNA-PKcs; Defect; Development; Double Strand Break Repair; Elements; Fostering; Gene Targeting; Gene Therapy Agent; Genome; Genomic Instability; Human; Human Genome; In Vitro; Ligase; Lysine; Maintenance; Malignant Neoplasms; Methyl Methanesulfonate; Methyltransferase; Modification; Mutagenesis; NBS1 gene; Neoplasm Metastasis; Nonhomologous DNA End Joining; Phase; Phosphorylation; Phosphotransferases; Plasmids; Process; Proliferating; Proteins; Role; Serine; Site; Small Interfering RNA; Stress; System; Testing; Tissues; Transposase; Viral; XRCC4 gene; base; cancer initiation; cancer radiation therapy; cancer therapy; cell growth; chemotherapy; endodeoxyribonuclease SceI; homologous recombination; human disease; human tissue; hydroxyurea; in vivo; inhibitor/antagonist; insight; leukemia/lymphoma; nuclease; public health relevance; treatment strategy; viral DNA

DESCRIPTION (provided by applicant): Metnase is a human protein with a SET (lysine methylase) domain and a Mariner transposase (nuclease) domain. Metnase is implicated in several aspects of DNA dynamics. Metnase promotes integration of DNA in a sequence-independent manner, but it is not known if integration sites are random. Metnase interacts with DNA ligase IV (LigIV) and NBS1, and appears to be an alternative to the well-known LigIV binding partner, XRCC4. LigIV and XRCC4 function in the final step of DNA double-strand break (DSB) repair by non-homologous end-joining (NHEJ). Metnase increases the efficiency and accuracy of NHEJ of plasmid substrates, and therefore appears to augment, or function redundantly with, classical NHEJ factors. Both SET and nuclease domains are required to promote NHEJ. Metnase has no apparent role in DSB repair by homologous recombination, but siRNA knockdown of Metnase suppresses random integration and enhances homology-directed integration (gene targeting). Metnase is not an active transposase as it does not efficiently mobilize endogenous Mariner elements. However, Metnase influences translocations perhaps reflecting its role in NHEJ. Defects in classical NHEJ proteins cause genome instability and predispose to cancer. Metnase is expressed in most human tissues, and Metnase levels are generally highest in proliferating tissues. siRNA knockdown of Metnase slows cell growth by elongating S phase, and sensitizes cells to replication stress induced by hydroxyurea and methylmethane sulfonate. Metnase is phosphorylated after DNA damage on serine 495 (S495), but the responsible kinase is unknown. Metnase interacts with TopoII1 and promotes TopoII1 chromosome decatenation activity. TopoII1 has been implicated in chromosomal translocations, including chemotherapy-induced translocations in secondary tumors. Our central hypothesis is that Metnase influences genome integrity through its roles in NHEJ, DNA integration, and chromosomal translocation. Much of what is currently known about Metnase is based on in vitro and plasmid-based in vivo assays. Here we propose two Specific Aims focused on in vivo chromosomal endpoints that will define the functional significance of the Metnase SET, nuclease, and phosphorylation domains in NHEJ and DNA integration. We will also determine the functional significance of the Metnase-LigIV interaction in NHEJ and integration, and whether Metnase influences chromosome translocations when TopoII1 is inhibited. These projects will provide mechanistic information about Metnase function during chromosomal DSB repair, integration and translocation. This information will provide new insights into (i) cellular stress responses and the maintenance of genome integrity, which relate to cancer etiology and treatment strategies; and (ii) the machinery responsible for DNA integration, which directly regulates genome modification by viral and non-viral DNA insertion, and may also be important for chromosomal translocations in human diseases including leukemias and lymphomas. Mechanistic insights into these processes will foster the development of more effective and safer cancer radio- and chemotherapy protocols, anti-viral agents, and gene therapy systems. PUBLIC HEALTH RELEVANCE: The human protein Metnase functions in DNA double-strand break repair, DNA integration into the human genome, and chromosomal translocations. The proposed studies will provide mechanistic information about cellular functions of Metnase. This information will provide new insights into cellular stress responses and the maintenance of genome integrity, both of which are important for cancer initiation and progression, and for cancer treatment. The proposed studies are also relevant to mechanisms of genome modification (mutagenesis) by viral and non-viral DNA insertion, and chromosomal translocations in human diseases including leukemias and lymphomas. Mechanistic insights into these processes will foster development of more effective and safer cancer radio- and chemotherapy protocols, anti-viral agents, and gene therapy systems.

描述（由申请人提供）： Metnase 是一种具有 SET（赖氨酸甲基化酶）结构域和 Mariner 转座酶（核酸酶）结构域的人类蛋白质。 Metnase 与 DNA 动力学的多个方面有关。 Metnase 以与序列无关的方式促进 DNA 整合，但尚不清楚整合位点是否是随机的。 Metnase 与 DNA 连接酶 IV (LigIV) 和 NBS1 相互作用，似乎是众所周知的 LigIV 结合伴侣 XRCC4 的替代品。 LigIV 和 XRCC4 在通过非同源末端连接 (NHEJ) 修复 DNA 双链断裂 (DSB) 的最后一步中发挥作用。 Metnase 提高了质粒底物 NHEJ 的效率和准确性，因此似乎增强了经典 NHEJ 因子，或与经典 NHEJ 因子一起发挥冗余作用。 SET 和核酸酶结构域都是促进 NHEJ 所必需的。 Metnase 在同源重组 DSB 修复中没有明显作用，但 Metnase 的 siRNA 敲低可抑制随机整合并增强同源定向整合（基因靶向）。 Metnase 不是活性转座酶，因为它不能有效地动员内源性 Mariner 元件。然而，Metnase 对易位的影响可能反映了它在 NHEJ 中的作用。经典 NHEJ 蛋白的缺陷会导致基因组不稳定并易患癌症。 Metnase 在大多数人体组织中表达，并且 Metnase 水平通常在增殖组织中最高。 Metnase 的 siRNA 敲低可通过延长 S 期来减缓细胞生长，并使细胞对羟基脲和甲基甲磺酸盐诱导的复制应激敏感。 DNA 丝氨酸 495 (S495) 损伤后，Metnase 被磷酸化，但负责的激酶尚不清楚。 Metnase 与 TopoII1 相互作用并促进 TopoII1 染色体去连接活性。 TopoII1 与染色体易位有关，包括继发性肿瘤中化疗诱导的易位。我们的中心假设是 Metnase 通过其在 NHEJ、DNA 整合和染色体易位中的作用影响基因组完整性。目前对 Metnase 的了解大多基于体外和基于质粒的体内测定。在这里，我们提出了两个专注于体内染色体终点的具体目标，这些目标将定义 Metnase SET、核酸酶和磷酸化结构域在 NHEJ 和 DNA 整合中的功能意义。我们还将确定 Metnase-LigIV 相互作用在 NHEJ 和整合中的功能意义，以及当 TopoII1 被抑制时 Metnase 是否影响染色体易位。这些项目将提供有关染色体 DSB 修复、整合和易位过程中 Metnase 功能的机制信息。这些信息将为（i）细胞应激反应和基因组完整性的维护提供新的见解，这与癌症病因学和治疗策略有关； (ii) 负责 DNA 整合的机制，它通过病毒和非病毒 DNA 插入直接调节基因组修饰，并且对于包括白血病和淋巴瘤在内的人类疾病中的染色体易位也可能很重要。对这些过程的机制见解将促进更有效、更安全的癌症放射和化疗方案、抗病毒药物和基因治疗系统的开发。 公共卫生相关性： 人类蛋白质 Metnase 在 DNA 双链断裂修复、DNA 整合到人类基因组中以及染色体易位中发挥作用。拟议的研究将提供有关 Metnase 细胞功能的机制信息。这些信息将为细胞应激反应和基因组完整性的维持提供新的见解，这两者对于癌症的发生和进展以及癌症治疗都很重要。拟议的研究还涉及通过病毒和非病毒 DNA 插入进行基因组修饰（诱变）的机制，以及人类疾病（包括白血病和淋巴瘤）中的染色体易位。对这些过程的机制洞察将促进更有效、更安全的癌症放疗和化疗方案、抗病毒药物和基因治疗系统的开发。