Characterization and testing of novel genes in DNA double-strand break repair

描述（由申请人提供）：DNA双链断裂（DSB）对细胞具有高度毒性，可通过电离辐射（IR）或链间交联剂引入，但也可在DNA复制过程中自发发生。如果错误修复，DSB可能导致细胞死亡，突变和癌症。此外，DSB修复（DSBR）缺陷是许多其他人类疾病的基础，包括与放射敏感性，免疫功能障碍，神经变性和过早衰老相关的疾病。因此，重要的是确定所有参与DSBR的基因。 在人类细胞中，在过去的十年中，许多参与DSBR的基因已经被鉴定出来，通常是基于与低等真核生物（如酵母）中相应直系同源物的序列同源性。然而，最近已经变得明显的是，DSBR途径在人类中比在低等真核生物中复杂得多，并且低等真核生物并不编码参与人类DSBR的所有蛋白质。 通过对3,600多个人类双色微阵列数据集进行全局元分析（GMA），结合文献数据挖掘软件，开发了一种基于实验间行为预测未知基因功能的新方法。基于这种方法，一个新的人类和明显的脊椎动物特异性基因，暂命名为DNARR 1（DNA修复相关1），已被预测和确定发挥作用的DSBR。这种作用不能根据序列同源性来预测。强有力的实验证据表明，DNARR 1与乳腺癌易感基因BRCA 1和BRCA 2一样，通过同源重组参与DSBR，这是一种具有已知肿瘤抑制功能的重要途径。由于DNARR 1位于染色体9q21.13，这是几种类型癌症中等位基因不平衡的区域，因此该基因与许多其他HRR基因一样，也可能代表新的癌症易感性位点。在本提案的目的1中，将研究DNARR 1在同源重组DNA修复（HRR）中的作用。特别地，将确定DNARR 1是否在IR诱导的RAD 51病灶形成之前或之后起作用，DNARR 1是否与DNA结合以及DNARR 1与哪些HRR蛋白相互作用。 使用GMA来鉴定潜在的DSBR基因，DNARR 1是迄今为止测试的唯一基因。然而，GMA已经预测了15个其他新的DSBR基因，这些基因已经缩小到我们认为最有可能参与DSBR的六个基因。在目标2中，将使用体外遗传破坏方法研究这六个基因在DSBR中的作用。具体而言，将检测这六种基因中任何一种的功能缺失是否会使人细胞对IR（即非同源末端连接）或干扰DNA复制的药物（即HRR）敏感。对于在这六个基因中鉴定的任何新的DSBR基因，将确定蛋白质相互作用伴侣，并测试与DNA结合的能力，以进一步确定其在DSBR中的作用。这项研究基于一种独特的预测基因功能的方法，将扩大我们对人类DSBR相关基因的了解，并确定新的疾病易感性候选者。 公共卫生相关性：DNA损伤是正常人类细胞中相当频繁的事件，其具有在需要时修复这种损伤的机制，即使当损伤严重到足以通过双链断裂（DSB）将DNA切割成两半时，并且DSB修复中的缺陷是许多人类疾病的原因，最常见的是癌症的易感性，但也是放射敏感性，免疫功能障碍，神经退化和过早衰老。在这里，一种新的计算方法预测了7个新的人类基因参与DSB修复，通过整合大规模文献数据挖掘的共表达模式的荟萃分析。我们已经通过同源重组（HR）验证了这些基因中的第一个在BRCA 1/2相关DSB修复中的作用，并且在本资助提案中，将进一步确定该基因在HR中的确切作用，以及测试和进一步研究其他六个基因在DSB修复中的作用。

DESCRIPTION (provided by applicant): DNA double-strand breaks (DSBs) are highly toxic to cells, and can be introduced by ionizing radiation (IR) or interstrand crosslinking agents, but also occur spontaneously during DNA replication. If mis-repaired, DSBs can result in cell death, mutations and cancer. In addition, defects in DSB repair (DSBR) underpin many other human diseases, including disorders associated with radiosensitivity, immune dysfunction, neurodegeneration and premature aging. Therefore, it is important to define all of the genes involved in DSBR. In human cells, many of the genes involved in DSBR have been identified during the last decade, frequently based on sequence homology to their respective orthologs in lower eukaryotes, such as yeast. Recently, however, it has become evident that DSBR pathways are much more complex in humans than in lower eukaryotes, and that lower eukaryotes do not encode all of the proteins involved in human DSBR. A new method has been developed to predict unknown gene function on the basis of inter-experimental behavior by using a global meta-analysis (GMA) of over 3,600 human 2-color microarray datasets in combination with literature data-mining software. Based on this approach, a novel human and apparently vertebrate-specific gene, tentatively named DNARR1 (DNA Repair Related 1), has been predicted and determined to play a role in DSBR. Such a role could not have been predicted on the basis of sequence homology. Strong experimental evidence suggests that DNARR1, like the breast cancer susceptibility genes BRCA1 and BRCA2, is involved in DSBR by homologous recombination, an essential pathway with known tumor-suppressor function. Since DNARR1 is located chromosome 9q21.13, a region of allelic imbalance in several types of cancers, this gene, like many other HRR genes, may also represent a new cancer susceptibility locus. In Aim 1 of this proposal the role of DNARR1 in homologous recombinational DNA repair (HRR) will be investigated. In particular, it will be determined if DNARR1 functions before or after IR-induced RAD51 focus formation, if DNARR1 binds to DNA and which HRR proteins DNARR1 interacts with. Using GMA to identify potential DSBR genes, DNARR1 is the only gene tested so far. However, the GMA has predicted 15 other novel DSBR genes, and these have been narrowed down to the six that we believe have the highest potential to be involved in DSBR. In Aim 2 these six genes will be investigated for their role in DSBR using in-vitro methods of genetic disruption. Specifically, it will be tested if functional loss of any of these six genes sensitizes human cells to IR (i.e. non-homologous end-joining) or drugs that interfere with DNA replication (i.e. HRR). For any new DSBR gene identified among these six, protein interaction partners will be determined and the ability to bind to DNA will be tested to further define its role in DSBR. This research, based on a unique approach to predict gene function, will expand our knowledge of human DSBR- associated genes and identify new disease susceptibility candidates. PUBLIC HEALTH RELEVANCE: DNA damage is a fairly frequent event in normal human cells, that have mechanisms in place to repair this damage when needed, even when the damage is severe enough to cleave the DNA in half via a double- stranded break (DSB), and defects in the repair of DSBs are responsible for many human diseases, the most frequent being a predisposition to cancer, but also radiosensitivity, immune dysfunction, neurodegeneration and premature aging. Here, a novel computational approach has predicted the involvement of seven new human genes in DSB repair by integrating a meta-analysis of co-expression patterns with large-scale literature data-mining. We have validated the role for the first of these genes in BRCA1/2-related DSB repair by homologous recombination (HR), and, in this grant proposal, will further define the exact role of this gene in HR, as well as test and further study the role of the other six genes in DSB repair.