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是一种具有集合（赖氨酸甲基酶）结构域的人蛋白和水手转座酶（核酸酶）结构域。 MetNase与DNA动力学的多个方面有关。 metnase以序列无关的方式促进了DNA的整合，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，并且似乎是众所周知的联络结合伴侣XRCC4的替代方法。在DNA双链断裂（DSB）修复的最后一步中，LIGIV和XRCC4功能通过非同源末端连接（NHEJ）。 metnase提高了质粒底物NHEJ的效率和准确性，因此似乎会增强或与经典的NHEJ因子相互作用。促进NHEJ都需要集合和核酸酶结构域。 MetNase通过同源重组在DSB修复中没有明显的作用，但是Metnase的siRNA敲低抑制了随机整合并增强了同源指导的整合（基因靶向）。 MetNase不是活性转座酶，因为它不能有效动员内源性水手元素。但是，metnase可能会影响易位反映其在NHEJ中的作用。经典NHEJ蛋白的缺陷会导致基因组不稳定性和易感性癌症。 metnase在大多数人体组织中表达，而在增殖组织中，metnase水平通常最高。 metnase的siRNA敲低通过伸长S相减慢细胞的生长，并使细胞对羟基脲和甲基甲烷磺酸盐诱导的复制应激敏感。在丝氨酸495（S495）上DNA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染色体衰减活性。 topoii1与染色体易位有关，包括化学疗法诱导的继发肿瘤的易位。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来影响基因组完整性。目前，关于MetNase的许多知识都是基于体内和基于质粒的体内测定的。在这里，我们提出了两个特定目的，这些特定目的集中在体内染色体终点，这些终端将定义NHEJ和DNA整合中MetNase集，核酸酶和磷酸化域的功能意义。我们还将确定NHEJ和整合中Metnase-ligiv相互作用的功能意义，以及当抑制topoii1时，Metnase是否会影响染色体易位。这些项目将在DSB修复，整合和易位期间提供有关Metnase功能的机械信息。该信息将为（i）细胞应激反应和基因组完整性的维持提供新的见解，这些疾病与癌症的病因和治疗策略有关； （ii）负责DNA整合的机械，该机械直接通过病毒和非病毒DNA插入来调节基因组修饰，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。对这些过程的机理见解将促进更有效，更安全的癌症放射和化学疗法方案，抗病毒药物和基因治疗系统的发展。 公共卫生相关性： 人蛋白元酶在DNA双链断裂修复，DNA整合到人基因组中的作用和染色体易位。拟议的研究将提供有关MetNase细胞功能的机械信息。该信息将为细胞应力反应和基因组完整性的维持提供新的见解，这对癌症的启动和进展以及癌症治疗都很重要。拟议的研究还与病毒和非病毒DNA插入的基因组修饰机制（诱变）以及人类疾病中的染色体易位有关，包括白血病和淋巴瘤。对这些过程的机械洞察力将促进开发更有效，更安全的癌症放射和化学疗法方案，抗病毒药物以及基因治疗系统。