METNASE ROLES IN NHEJ, DNA INTEGRATION AND TRANSLOCATION

描述（由申请人提供）： Metnase是具有SET（赖氨酸甲基化酶）结构域和Mariner转座酶（核酸酶）结构域的人蛋白质。Metnase涉及DNA动力学的几个方面。Metnase以不依赖于序列的方式促进DNA的整合，但不知道整合位点是否是随机的。Metnase与DNA连接酶IV（LigIV）和NBS 1相互作用，似乎是众所周知的LigIV结合伴侣XRCC 4的替代品。LigIV和XRCC 4通过非同源末端连接（NHEJ）在DNA双链断裂（DSB）修复的最后步骤中发挥作用。Metnase增加了质粒底物的NHEJ的效率和准确性，因此似乎增加了经典NHEJ因子或与经典NHEJ因子冗余地起作用。SET和核酸酶结构域都是促进NHEJ所必需的。Metnase在通过同源重组的DSB修复中没有明显的作用，但是Metnase的siRNA敲低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影响易位，可能反映了其在NHEJ中的作用。经典NHEJ蛋白的缺陷导致基因组不稳定并易患癌症。Metnase在大多数人组织中表达，并且Metnase水平通常在增殖组织中最高。siRNA敲除Metnase通过延长S期减缓细胞生长，并使细胞对由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）上的DNA损伤后，Metnase被磷酸化，但负责的激酶是未知的。Metnase与TopoII 1相互作用并促进TopoII 1染色体脱连锁活性。TopoII 1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。目前关于Metnase的许多已知的是基于体外和基于质粒的体内测定。在这里，我们提出了两个具体的目标集中在体内染色体终点，将定义的Metnase SET，核酸酶，磷酸化结构域在NHEJ和DNA整合的功能意义。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TopoII 1被抑制时Metnase是否影响染色体易位。这些项目将提供有关Metnase在染色体DSB修复、整合和易位过程中功能的机制信息。这些信息将提供新的见解（i）细胞应激反应和基因组完整性的维持，这与癌症病因学和治疗策略有关;和（ii）负责DNA整合的机制，其通过病毒和非病毒DNA插入直接调节基因组修饰，并且可能对人类疾病（包括白血病和淋巴瘤）中的染色体易位也很重要。对这些过程的机制性见解将促进更有效和更安全的癌症放射和化学治疗方案，抗病毒药物和基因治疗系统的发展。 公共卫生相关性： 人类蛋白质Metnase在DNA双链断裂修复、DNA整合到人类基因组和染色体易位中发挥作用。这些研究将为Metnase的细胞功能提供机制信息。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发展以及癌症治疗都很重要。拟议的研究还涉及病毒和非病毒DNA插入引起的基因组修饰（诱变）机制，以及人类疾病（包括白血病和淋巴瘤）中的染色体易位。对这些过程的机制性见解将促进更有效和更安全的癌症放射和化学治疗方案，抗病毒药物和基因治疗系统的发展。

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.