METNASE ROLES IN NHEJ, DNA INTEGRATION AND TRANSLOCATION

描述(由申请人提供)： Metnase是一种含有SET(赖氨酸甲基酶)结构域和Mariner转座酶(核酸酶)结构域的人类蛋白质。Metnase与DNA动力学的几个方面有关。Metnase以一种不依赖于序列的方式促进DNA的整合，但整合位点是否随机尚不清楚。Metnase与DNA连接酶IV(LigIV)和NBS1相互作用，似乎是众所周知的LigIV结合伙伴XRCC4的替代品。LigIV和XRCC4在DNA双链断裂(DSB)非同源末端连接(NHEJ)修复的最后一步发挥作用。Metnase提高了质粒底物的NHEJ的效率和准确性，因此似乎增加了经典的NHEJ因子，或与经典的NHEJ因子冗余地发挥作用。SET结构域和核酸酶结构域都需要促进NHEJ。Metnase在通过同源重组修复DSB中没有明显的作用，但Metnase的siRNA敲除抑制了随机整合，并增强了同源定向整合(基因打靶)。Metnase不是一种活性转座酶，因为它不能有效地动员内源水手元件。然而，Metnase影响易位，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都有表达，而且在增殖组织中的水平通常最高。Metnase的siRNA敲除通过延长S时相来减缓细胞的生长，并使细胞对羟基脲和甲基甲烷磺酸诱导的复制应激敏感。丝氨酸495(S495)上的DNA损伤后，金属酶被磷酸化，但负责的激酶尚不清楚。Metnase与TopOII1相互作用，促进TopOII1染色体的破坏活性。Topo II1与染色体易位有关，包括化疗引起的继发性肿瘤易位。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的完整性。目前对Metnase的大部分了解都是基于体外和基于体内质粒的检测。在这里，我们提出了两个特定的目标，集中在体内的染色体终点，将定义偏位酶集，核酸酶，和磷酸化结构域在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、整合和易位过程中代谢酶功能的机械性信息。这些信息将为以下方面提供新的见解：(I)与癌症病因和治疗策略有关的细胞应激反应和基因组完整性的维持；以及(Ii)负责DNA整合的机制，该机制通过病毒和非病毒DNA插入直接调节基因组修改，并可能对包括白血病和淋巴瘤在内的人类疾病的染色体易位起重要作用。对这些过程的机械性见解将促进更有效和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发展。 公共卫生相关性： 人类蛋白质代谢酶在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。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，这两者对癌症的发生和发展以及癌症治疗都是重要的。拟议的研究还涉及病毒和非病毒DNA插入的基因组修改(突变)机制，以及包括白血病和淋巴瘤在内的人类疾病中的染色体易位。对这些过程的机械性见解将促进更有效和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。

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.