Basic study on the mechanisms of carcinogenesis and aging with special reference to the function of RecQ family helicases

在这项研究中，我们试图阐明RecQ家族解旋酶的功能，以了解由于RecQ家族解旋酶的缺陷而导致的基因组不稳定性导致癌症易感性和过早衰老的原因，并通过使用易于用于遗传分析的芽殖酵母和鸡DT40细胞以及培养的人类细胞获得以下结果。通过对BLm-/-和BLm-/-/RAD54-/-DT40细胞的产生和分析表明，在BLm-/-和BLm-/-/RAD54-/-DT40细胞中，大部分增加的SCE是通过同源重组和BLm功能来减少DNA复制过程中DNA双链断裂的形成。Werner综合征基因产物WRN与Ubc9和SUMO-1相互作用，实际上与SUMO-1.3结合。我们发现的一种新的Werner解旋酶相互作用蛋白WHIP与WRN共定位在细胞核中，并且这些蛋白的相互作用需要ATP，这表明这些蛋白是动态相互作用的。利用酵母突变体进行的遗传分析表明，在DNA聚合酶δ、rfc和增殖细胞核抗原的突变体中，WHIP基因的过度表达导致了细胞的死亡，表明该突变体与DNA聚合酶δ、rfc和增殖细胞核抗原之间存在功能关系。此外，酵母-双杂交系统还表明了WHIP与δ聚合酶之间的物理相互作用。酵母菌分析还表明，酵母BLM/WRN同源物SGS1与参与DNA复制启动的Mre11、SLD3和SLD5存在遗传相互作用。

In this study, we tried to clarify the function of RecQ family helicases to understand the genomic instability resulting in predisposition to cancer and premature aging due to the defect in RecQ family helicases, and obtained following results by using budding yeasts and chicken DT40 cells, which are easily applicable to genetic analysis, as well as cultured human cells.1. It has been indicated by generating and analyzing BLM-/- and BLM-/-/RAD54-/- DT40 cells that the majority of increased SCEs in BLM disrupted cells are formed via homologous recombination and BLM functions to decrease the formation of DNA double strand breaks during DNA replication.2. Werner syndrome gene product, WRN, interacted with Ubc9 and SUMO-1 and was actually conjugated with SUMO-1.3. A novel Werner helicase interacting protein, WHIP, which we found, was co-localized with WRN in the nuclei, and the interaction of these proteins required ATP, indicating a dynamic interaction of these proteins.4. The genetic analyzes using yeast mutants that overexpression of WHIP in mutants of DNA polymerase δ, RFC, or PCNA resulted in the lethality of the cells indicated the existence of functional relationship between WHIP and DNA polymerase δ, RFC, and PCNA. In addition, physical interaction between WHIP and DNA polymerase δ was indicated by the yeast-two hybrid system.5. Analyzes using yeasts also indicated the genetical interaction of the yeast BLM/WRN homologue, Sgs1 with Mre11, Sld3, and Sld5 which are involved in the initiation of DNA replication.