L2DTL Function in Maintaining Genomic Stability during Drosophila Development

描述(由申请人提供)：DNA复制受到严格控制，每个细胞周期只允许一轮。过度复制可能导致严重的后果，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有助于将复制型解旋酶复合体装载到复制起点上，其正确的表达和及时的破坏是S阶段“一次且唯一”复制控制的重要组成部分。值得注意的是，cdt1/DUP及其抑制物Ginin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家族的成员在维持基因组稳定性方面起着至关重要的作用，在S阶段进入后立即调节CDT1/DUP的水平。特别是，致死2无牙(L2DTL)作为一个特异性因子，将CDT1/DUP靶向含有cullin 4(CUL4)和DNA损伤结合因子1(DDB1)的E3泛素连接酶(CUL4)，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：肝脏是为数不多的维持再复制细胞周期的成人组织之一。值得注意的是，果蝇的研究强调了CDT/DUP在经历再复制周期的组织中的作用。由于CDT1/DUP在不同的细胞和发育环境中已经在果蝇中得到了广泛的研究，黑腹果蝇成为阐明L2DTL家族在调节CDT1/DUP水平和维持基因组稳定性方面的一个很好的模型。为了确定L2DTL在多细胞生物体中的功能，这项建议将集中在鉴定和表征具有L(2)DTL基因突变的苍蝇种群。特征将包括使用BrdU掺入分析来评估胚胎组织中的再复制表型，以及使用间接免疫荧光(特定目标1)来评估CDT1/DUP水平。此外，将产生抗L2DTL抗体，以研究L2DTL蛋白在经历有丝分裂和内周期细胞周期的细胞中的表达模式和亚细胞定位。最后，由于L(2)DTL基因突变导致胚胎死亡，将建立诱导幼虫和成体组织有丝分裂克隆的果蝇系，以阐明L2DTL在维持体细胞组织基因组稳定性方面的作用，通过BrdU掺入和Cdt1/DUP积累(特异性目标2)。实验结果应该支持这样的假设，即L2DTL对于防止在所有细胞环境中的重复复制是必不可少的。

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.