L2DTL Function in Maintaining Genomic Stability during Drosophila Development

描述（由申请人提供）：DNA复制受到严格调控，每个细胞周期仅允许一轮。过度复制可导致严重后果，包括基因组不稳定、癌症或死亡。因此，Cdt 1/Dup复制许可因子的水平受到严格调控。Cdt 1/Dup有助于将复制解旋酶复合物加载到复制起点上，其正确表达和及时破坏是S期“一次性”复制控制的重要组成部分。值得注意的是，Cdt 1/Dup及其抑制剂Geminin水平升高与越来越多的人类癌症相关。最近的报道表明，L2 DTL家族的成员在维持基因组的稳定性发挥了至关重要的作用，通过调节Cdt 1/Dup水平后立即进入S期。特别是，L2 DTL作为特异性因子，将Cdt 1/Dup靶向含有Cullin 4（Cul 4）和DNA损伤结合因子1（DDB 1）的E3泛素连接酶，用于随后的蛋白酶体降解。L2 DTL水平的增加本身与肝细胞癌相关：肝脏是维持再复制细胞周期的少数成人组织之一。值得注意的是，果蝇的研究突出了Cdt/Dup在经历再复制周期的组织中的作用。由于Cdt 1/Dup已在果蝇的各种细胞和发育背景下进行了广泛的研究，果蝇作为一个很好的模型，阐明L2 DTL家族在调节Cdt 1/Dup水平和维持基因组稳定性的作用。为了确定L2 DTL在多细胞生物体中的功能，该提议将集中于鉴定和表征在l（2）dtl基因中具有突变的蝇类。表征将包括使用BrdU掺入分析评估胚胎组织中的再复制表型，并使用间接免疫荧光评价Cdt 1/Dup水平（具体目的1）。此外，将产生抗L2 DTL抗体以研究L2 DTL蛋白表达模式和经历有丝分裂和内循环细胞周期的细胞中的亚细胞定位。最后，由于l（2）dtl基因的突变导致胚胎致死，将产生用于在幼虫和成虫组织中诱导有丝分裂克隆的果蝇系，以阐明L2 DTL在维持体细胞组织中基因组稳定性中的作用，如通过BrdU掺入和Cdt 1/Dup积累所测量的（具体目标2）。实验结果应该支持这一假设，即L2 DTL是必不可少的，以防止在所有的细胞环境中的再复制。

DESCRIPTION (provided by applicant): DNA replication is strictly regulated to allow only one round per cell cycle. Over-replication can lead to serious consequences including genomic instability, cancer, or death. Thus, levels of the Cdt1/Dup replication licensing factor are tightly regulated. Cdt1/Dup helps load the replicative helicase complex onto origins of replication, and its proper expression and timely destruction are an important part of the "once and only" replication control during S phase. Significantly, elevated levels of Cdt1/Dup and its inhibitor Geminin are associated with an increasing number of human cancers. Recent reports indicate that members of the L2DTL family play a vital role in maintaining genomic stability by modulating Cdt1/Dup levels immediately after S phase entry. In particular, Lethal 2 Denticleless (L2DTL) acts as a specificity factor, targeting Cdt1/Dup to the E3 ubiquitin ligase containing Cullin 4 (Cul4) and DNA damage binding factor 1 (DDB1) for subsequent proteasome degradation. Increased levels of L2DTL itself are associated with hepatocellular carcinomas: the liver being one of the few adult tissues sustaining a re-replicative cell cycle. Notably, Drosophila studies highlight the role of Cdt/Dup in tissues undergoing re-replicative cycles. Since Cdt1/Dup has been extensively studied in Drosophila in various cell and development contexts, Drosophila melanogaster serves as an excellent model for elucidating the role of the L2DTL family in regulating Cdt1/Dup levels and maintaining genomic stability. To determine the function of L2DTL in a multicellular organism, this proposal will focus on identifying and characterizing fly stocks with mutations in the l(2)dtl gene. Characterization will include an assessment of the re-replication phenotype in embryonic tissues using BrdU incorporation analysis and evaluation of Cdt1/Dup levels using indirect immunofluorescence (specific aim 1). Further, anti-L2DTL antibodies will be generated to investigate the L2DTL protein expression pattern and subcellular localization in cells undergoing mitotic and endocyclic cell cycles. Finally, since mutation of the l(2)dtl gene results in embryonic lethality, Drosophila lines for inducing mitotic clones in larval and adult tissues will be generated to elucidate the role of L2DTL in maintaining genomic stability in somatic tissues as measured by BrdU incorporation and Cdt1/Dup accumulation (specific aim 2). Experiment results should support the hypothesis that L2DTL is essential for preventing re-replication in all cell contexts.