Isolation and Analysis of Human DNA Repair Genes

描述（由申请人提供）：SNM1（对氮芥的敏感性）最初在酿酒葡萄球菌中被鉴定为具有对链间交联剂抗性的基因，但对其他形式的DNA损伤没有抗性。在哺乳动物细胞中，SNM1基因家族中有五个已确定的成员，本申请建议继续我们对其中两个基因SNM1和SNM1B的研究。我们的初步研究结果表明，这两种蛋白质与DNA-PK相互作用，正如其他被称为Artemis的家族成员所显示的那样。此外，我们发现SNM1是延迟进入中期的有丝分裂应激检查点的一个组成部分。这种新的检查点是最近由人类Chfr基因的特征定义的，不同于由Mad和Bub蛋白定义的纺锤体检查点。与在有丝分裂检查点中的作用一致，我们发现SNM1和SNM1B在双杂交筛选中都与有丝分裂纺锤体蛋白Astrin相互作用，并且SNM1与后期促进复合物的组分共同免疫沉淀。最后，我们在小鼠中破坏了SNMI，并在纯合子和杂合子动物中观察到加速的肿瘤发生和肥胖表型。本研究的目标是研究SNM1和SNM1B在NHEJ和DNA损伤诱导的细胞周期检查点中的可能作用，进一步研究这些蛋白在有丝分裂应激检查点中的功能，并在小鼠中建立这些基因的敲除模型，作为阐明其肿瘤抑制功能的方法。

DESCRIPTION (provided by applicant): SNM1 (for sensitivity to nitrogen mustard) was originally identified in S. cerevisiae as a gene that conferred resistance to interstrand cross-linking agents, but not to other forms of DNA damage. In mammalian cells there are five identified members of the SNM1 gene family, and this application proposes to continue our studies on two of these genes termed SNM1 and SNM1B. Our preliminary findings show that both proteins interact with DNA-PK as has been shown by others with another member of the family termed Artemis. In addition, we have found that SNM1 is a component of a mitotic stress checkpoint that delays entry into metaphase. This novel checkpoint was recently defined by the characterization of the human Chfr gene, and is distinct from the well-characterized spindle checkpoint defined by the Mad and Bub proteins. Consistent with a role in a mitotic checkpoint, we have found that both SNM1 and SNM1B interact with the mitotic spindle protein Astrin in a two-hybrid screen, and that SNM1 co-immunoprecipitates with components of the anaphase-promoting complex. Finally, we have disrupted SNMI in the mouse and have observed both accelerated tumorigenesis and obesity phenotypes in the homozygous and heterozygous animals. The goals of this proposal are to examine the possible roles of SNM1 and SNM1B in NHEJ and DNA damage-induced cell cycle checkpoints, to further examine the function of these proteins in mitotic stress checkpoints, and to develop additional knockout models of these genes in mice as method to elucidate their function in tumor suppression.