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

描述(申请人提供)：DNA双链断裂(DSB)对细胞具有高度毒性，可由电离辐射(IR)或链间交联剂引入，但也可在DNA复制过程中自发发生。如果修复不当，DSB可能会导致细胞死亡、突变和癌症。此外，DSB修复缺陷(DSBR)是许多其他人类疾病的基础，包括与辐射敏感性、免疫功能障碍、神经退化和过早衰老相关的疾病。因此，确定DSBR中涉及的所有基因是很重要的。在人类细胞中，参与DSBR的许多基因在过去十年中已经被发现，通常基于它们在酵母等低等真核生物中各自的同源物的序列同源性。然而，最近有证据表明，人类的DSBR通路比低等真核生物复杂得多，低等真核生物并不编码人类DSBR所涉及的所有蛋白质。通过对3600多个人类双色微阵列数据集的全局荟萃分析(GMA)，结合文献数据挖掘软件，开发了一种基于实验间行为的未知基因功能预测的新方法。基于这种方法，一个新的人类和明显脊椎动物特有的基因，暂定为DNARR1(DNA修复相关1)，已经被预测并确定在DSBR中发挥作用。这种作用不可能在序列同源性的基础上被预测。强有力的实验证据表明，DNARR1与乳腺癌易感基因BRCA1和BRCA2一样，通过同源重组参与DSBR，这是一条具有已知肿瘤抑制功能的重要途径。由于DNARR1位于染色体9q21.13，这是几种癌症中等位基因失衡的区域，该基因与许多其他HRR基因一样，也可能代表一个新的癌症易感基因。在本提案的目标1中，将研究DNARR1在同源重组DNA修复(HRR)中的作用。特别是，将确定DNARR1是在IR诱导的RAD51焦点形成之前还是之后发挥功能，DNARR1是否与DNA结合，以及DNARR1与哪些HRR蛋白相互作用。使用GMA识别潜在的DSBR基因，DNARR1是到目前为止唯一检测到的基因。然而，GMA预测了另外15个新的DSBR基因，这些基因已经缩小到我们认为最有可能参与DSBR的6个基因。在目标2中，这六个基因将使用基因中断的体外方法来研究它们在DSBR中的作用。具体地说，将测试这六个基因中的任何一个的功能丧失是否会使人类细胞对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发现的任何新的DSBR基因，将确定蛋白质相互作用伙伴，并将测试其与DNA的结合能力，以进一步确定其在DSBR中的作用。这项研究基于一种预测基因功能的独特方法，将扩大我们对人类DSBR相关基因的知识，并确定新的疾病易感性候选者。 与公共卫生相关：DNA损伤在正常人类细胞中是相当频繁的事件，在需要时，细胞有修复这种损伤的机制，即使损伤足够严重，通过双链断裂(DSB)将DNA切割成两半，DSB修复缺陷导致许多人类疾病，最常见的是癌症易感性，但也包括辐射敏感性、免疫功能障碍、神经退化和过早衰老。在这里，一种新的计算方法通过将共表达模式的荟萃分析与大规模文献数据挖掘相结合，预测了七个新的人类基因参与DSB修复。我们已经通过同源重组(HR)验证了第一个基因在BRCA1/2相关DSB修复中的作用，并将在这项拨款提案中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.