Elucidating roles of RTEL in replication fork repair in Drosophila melanogaster

描述(申请人提供)：S期细胞的基因组特别容易受到脱氧核糖核酸的破坏，脱氧核糖核酸复制过程中出现的问题是基因组不稳定的一个重要来源，而基因组不稳定是癌症的一个重要标志。DNA解旋酶在对抗这些破坏稳定的事件中发挥着重要作用。强调这一点的事实是，许多解旋酶基因的突变与癌症易感性和早衰综合征有关，而这些患者的细胞具有高度的基因组不稳定性[1]。保守的DNA解旋酶基因Rtel中的SNPs和突变与多种癌症和各种形式的端粒不稳定性疾病先天性角化不良[2-5]有关[2-5]这项建议试图阐明Rtel解旋酶的DNA修复功能，最终目的是更好地理解与Rtel和其他DNA解旋酶的遗传缺陷相关的病理中的疾病发展机制。已经通过在患者细胞中的观察以及在小鼠和其他动物模型中的研究来探索RTE的功能，这些研究揭示了端粒长度维持的作用。此外，以前的研究表明，Rtel通过同源重组途径和复制叉处DNA交联链的修复，在对抗有丝分裂交叉中发挥作用，这可能导致杂合性丧失。尽管有这些发现，但小鼠模型的胚胎致死性和缺乏体内DNA修复试验限制了对rtel DNA修复作用的研究。由于果蝇端粒的非规范结构，除了许多可用的分子遗传学工具外，果蝇黑腹果蝇还是研究rtel功能的理想动物模型，这可能允许将基因组维持功能与端粒特异功能分开进行检查。这项研究的总体目标是阐明rtel在DNA修复中的生物学功能，并了解rtel如何保护DNA损伤的有害生物学后果，包括缺陷增殖和死亡。为此，这项建议的具体目标是(1)测试Rtel在同源重组中的功能模型，(2)测试Rtel在复制分叉进程中的功能模型，以及(3)确定Rtel在促进适当的细胞增殖、细胞周期进展和活性方面的作用。

DESCRIPTION (provided by applicant): The genomes of S-phase cells are especially vulnerable to DNA damage, and problems that arise during DNA replication are a significant source of genomic instability, an important hallmark of cancer. DNA helicases play an important role in counteracting these destabilizing events. This is emphasized by the fact that mutations in many of the helicase genes are associated with cancer predisposition and premature aging syndromes, and cells from these patients harbor a high level of genomic instability [1]. SNPs and mutations in the conserved DNA helicase gene RTEL have been associated with a variety of cancers and with forms of the telomere instability disorder dyskeratosis congenita [2-5] This proposal seeks to elucidate the DNA repair functions of the RTEL helicase with the ultimate goal of better understanding mechanisms of disease development in pathologies associated with genetic defects in RTEL and other DNA helicases. RTEL function has been explored through observations in patients' cells as well as studies in mouse and other animal models, which revealed roles in telomere length maintenance. In addition, previous studies have suggested roles for RTEL in counteracting mitotic crossovers, which can lead to loss of heterozygosity, through the homologous recombination pathway and in the repair of DNA crosslinks at replication forks. Despite these findings, the embryonic lethality of the mouse model and lack of in vivo DNA repair assays has limited the study of the DNA repair roles of RTEL. In addition to the many molecular genetic tools available, the fruit fly Drosophila melanogaster is an ideal animal model to study RTEL function due to the non-canonical structure of the fly telomeres, likely allowing for the examination of genome maintenance functions separately from the telomere- specific functions. The overall goal of this research is to elucidate RTEL biological function in DNA repair and understand how RTEL protects against the deleterious biological consequences of DNA damage including defective proliferation and death. To that end, the specific aims of this proposal are to (1) test models for RTEL function in homologous recombination, (2) test models for RTEL function in replication fork progression, and (3) determine the role of RTEL in promoting proper cellular proliferation, cell cycle progression, and viability.