L2DTL Function in Maintaining Genomic Stability during Drosophila Development

描述（由申请人提供）：DNA复制受到严格监管，每个细胞周期只允许一次复制。过度复制会导致严重的后果，包括基因组不稳定、癌症或死亡。因此，Cdt1/Dup复制许可因子的水平受到严格调控。Cdt1/Dup有助于将复制解旋酶复合体装载到复制起点上，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，Cdt1/Dup及其抑制剂Geminin水平的升高与人类癌症数量的增加有关。最近的报道表明，L2DTL家族成员在S期进入后立即调节Cdt1/Dup水平，在维持基因组稳定性方面起着至关重要的作用。特别是，致命2无牙（L2DTL）作为特异性因子，靶向Cdt1/ d1直至含有Cullin 4 （Cul4）和DNA损伤结合因子1 （DDB1）的E3泛素连接酶，用于随后的蛋白酶体降解。L2DTL水平升高本身与肝细胞癌有关：肝脏是维持细胞再生周期的少数成年组织之一。值得注意的是，果蝇研究强调了Cdt/Dup在经历再复制周期的组织中的作用。由于Cdt1/Dup已经在果蝇的各种细胞和发育背景下得到了广泛的研究，因此黑腹果蝇可以作为一个很好的模型来阐明L2DTL家族在调节Cdt1/Dup水平和维持基因组稳定性中的作用。为了确定L2DTL在多细胞生物中的功能，本研究将集中于鉴定和表征l(2)dtl基因突变的蝇群。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和使用间接免疫荧光评估Cdt1/Dup水平（特异性目的1）。此外，将产生抗L2DTL抗体，以研究L2DTL蛋白在有丝分裂和内环细胞周期中的表达模式和亚细胞定位。最后，由于l(2)dtl基因突变会导致胚胎致死性，因此将产生在幼虫和成体组织中诱导有丝分裂克隆的果蝇系，通过BrdU掺入和Cdt1/Dup积累来阐明L2DTL在维持体细胞组织基因组稳定性方面的作用（具体目的2）。实验结果应该支持L2DTL在所有细胞环境中对防止再复制至关重要的假设。

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.