Genome Instability in Cancer Development

传递遗传信息而不产生有害的遗传改变是最重要的任务之一。细胞已经进化出检查和修复潜在致命DNA损伤的系统。然而，当这些系统不能正常工作时，DNA损伤就会积累并导致遗传变化或细胞死亡。遗传变化的积累，被定义为基因组不稳定性，经常在包括癌症在内的各种类型的遗传疾病中观察到。基因组不稳定性已被证明是肿瘤抑制基因多重失活和原癌基因激活的前一步。在许多癌症中经常观察到的一种类型的基因组不稳定性是总染色体重排（GCR）。GCR包括易位、染色体臂缺失、间质缺失、倒位、扩增、染色体端-端融合和非整倍体。虽然对癌细胞中观察到的GCR的起源和机制知之甚少，但最近对遗传性癌症易感综合征中突变基因的研究已经开始证明，在DNA损伤反应，DNA修复和DNA重组中起作用的蛋白质在抑制自发和/或DNA损伤诱导的GCR中起着至关重要的作用。为了了解GCR的产生机制以及这种GCR的形成如何导致肿瘤发生，我们筛选了整个酵母基因组中增加GCR形成速率的突变或过表达。从突变筛选中选择了RAD 5和ELG 1，以进一步研究这些蛋白质保护基因组免受有害GCR形成的分子机制。 1.确定RAD 5直系同源物SHPRH的作用。 之前，我们在哺乳动物中鉴定了两个RAD 5直向同源物，并证明了RAD 5直向同源物SHPRH和HLTF通过辅助模板转换DNA损伤旁路机制来防止持续停滞的复制叉的崩溃，该机制使用姐妹染色单体的新生链进行损伤旁路的重组机制。在决定旁路机制的增殖细胞核抗原（PCNA）的不同修饰中，我们证明了PCNA被SHPRH和HLTF多泛素化。 虽然我们在体内检测了SHPRH和HLTF与肿瘤发生的关系，但这些基因的失活并没有导致肿瘤发生的增加。SHPRH具有一个独特的组蛋白相互作用结构域，称为PHD结构域。我们最近发现，这一领域是重要的SHPRH定位在核仁。在我们对调节PCNA泛素化的一般DNA损伤反应的表征中，我们意外地发现，我们用作DNA损伤反应对照的RPA 32的丝氨酸4和8的磷酸化依赖于DNA依赖性蛋白激酶。我们发现了SHPRH在核仁中的一种新功能，从而推动了该项目进入SHPRH在核仁中rRNA转录的新分子机制。我们发现SHPRH对rRNA转录的作用是mTOR依赖的。 2. ATAD 5（哺乳动物ELG 1同系物）：确定替代复制因子C（RFC）复合蛋白是否指导DNA修复途径和复制。 我们报道了Atad 5单倍不足的小鼠显示出高的肿瘤发生率。我们最近证实，胚胎7.5至8.5天是由ATAD 5的纯合无效突变引起的胚胎致死。此外，我们与NHGRI的Daphne Bells博士团队合作，在许多子宫内膜肿瘤中发现了ATAD 5基因的人体突变。我们还发现了几种罕见的多态性以及其他肿瘤类型的癌症突变。我们从NHGRI斑马鱼核心设施的TILLING项目中回收了一个斑马鱼atad 5无效等位基因。不像atad 5基因缺失的小鼠在胚胎期8.5天死亡，atad 5基因缺失的斑马鱼可以存活到受精后7天。atad 5基因敲除的斑马鱼受精卵经MMS处理后，其致死表型在孵化后即刻即明显。因此，我们相信，鉴定的ATAD 5变体的体内功能活性可以用atad 5缺失斑马鱼模型进行测试。 我们已经产生了各种异位表达的构建体与一个单一的序列变异体中发现的体细胞癌突变，以及在国际癌症基因组联盟的癌症基因组图谱使用定点诱变的数据库搜索。我们目前正在测试这些罕见的突变是否会影响ATAD 5的分子功能，并导致在小鼠和斑马鱼以及组织培养系统中观察到的表型。

Transmitting genetic information without creating deleterious genetic alterations is one of the most important tasks. Cells have evolved systems that check for and repair potentially lethal DNA damage. However, when these systems do not work properly, DNA damage accumulates and causes genetic changes or cell death. Accumulation of genetic changes, which is defined as a genomic instability is frequently observed in various types of genetic disorders including cancers. Genomic instability has been documented as a preceding step for multiple inactivations of tumor suppressor genes and activations of proto-oncogenes. One type of genomic instability observed frequently in many cancers is gross chromosomal rearrangement (GCR). GCR includes translocations, deletions of chromosome arm, interstitial deletions, inversions, amplifications, chromosome end-to-end fusion and aneuploidy. Although little is known about the origin and mechanisms of GCRs observed in cancer cells, recent studies on genes mutated in inherited cancer predisposition syndromes have started to demonstrate that proteins that function in DNA damage responses, DNA repair, and DNA recombination, play crucial roles in the suppression of spontaneous and/or DNA damage-induced GCRs. To understand mechanisms how GCRs are generated and how such GCR formation can lead tumorigenesis, we screened the entire yeast genome for mutations or overexpression that increase the rate of GCR formation. RAD5 and ELG1 from mutation screening were selected for further studies of molecular mechanisms of these proteins to protect genome from deleterious GCR formation. 1. Determine the role of RAD5 orthologs, SHPRH. Previously, we identified two RAD5 orthologs in mammals and demonstrated that RAD5 orthologs, SHPRH and HLTF function to prevent collapse of persistent stalled replication forks by assisting template switching DNA damage bypass mechanism that uses the nascent strand of the sister chromatid for recombination mechanism for damage bypass. Among different modifications of Proliferating Cell Nuclear Antigen (PCNA) that determine the bypass mechanisms, we demonstrated that PCNA is poly-ubiquitinated by SHPRH and HLTF. Although we tested for association of SHPRH and HLTF with tumorigenesis in vivo, the inactivation of these genes did not result in an increase in tumorigenesis. SHPRH has a unique histone interaction domain called PHD domain. We recently found that this domain is important for SHPRH localization in the nucleolus. In our characterization of the general DNA damage response regulating PCNA ubiquitination, we unexpectedly found that phosphorylation of Serine 4 and 8 of RPA32, which we used as a DNA damage response control, depends on DNA dependent protein kinase. We found a novel function of SHPRH in the nucleolus that drove the project into a novel molecular mechanism of SHPRH in rRNA transcription in the nucleolus. We found the function of SHPRH for rRNA transcription is mTOR-dependent. 2. ATAD5 (mammalian ELG1 homolog): determine whether alternative Replication Factor C (RFC) complex protein directs DNA repair pathways and replication. We reported that mice haploinsufficient in Atad5 showed a high incidence of tumorigenesis. We recently confirmed that embryonic day 7.5 to 8.5 as embryonic lethality caused by homozygous null mutation of ATAD5. In addition, in collaboration with Dr. Daphne Bells group in NHGRI, we found human somatic mutations of ATAD5 gene in many endometrial tumors. We also found several rare polymorphisms as well as cancer mutations in other tumor types. We recovered a zebrafish atad5 null allele from the TILLING project in the NHGRI zebrafish core facility. Unlike the atad5 null mouse, which dies at embryonic day 8.5, an atad5 null zebrafish survives until seven days post fertilization (dpf). The lethal phenotype of the atad5 null zebrafish became obvious right after hatching when fertilized eggs were treated with MMS. Therefore, we believe that in vivo functional activity of ATAD5 variants identified can be tested with the atad5 null zebrafish model. We have generated various ectopic expression constructs with a single sequence variant found in somatic cancer mutations as well as database search in the International Cancer Genome Consortium for the Cancer Genome Atlas using site-directed mutagenesis. We are currently testing whether these rare mutations found in affect ATAD5s molecular function and cause phenotypes observed in mice and zebrafish as well as in tissue culture system.