Gene Expression And Human Genetics

工作概述：为了剖析基因重组的生化步骤，我们选择关注一个关键的早期步骤：同源亲本dna之间的同源配对和链交换。同源重组的一个基本问题是如何在两个dna之间寻找同源性。在目前所有的模型中，同源重组蛋白，如典型的大肠杆菌RecA蛋白，装载到由一个双工DNA产生的单链DNA上，并扫描另一个双工形成突触（配对）复合体。最终，DNA链被交换，形成一个新的异双工。同源配对和链交换是由RecA及其真核同源物Rad51和Dmc1介导的两个亲本DNA之间最早的接触，同源重组是在DNA双链断裂（DSBs）时启动的。在出芽酵母（Saccharomyces cerevisae）减数分裂中催化DSB形成的蛋白质是SPO11基因的产物。该基因的破坏导致减数分裂停滞，孢子死亡和减数分裂重组缺乏。在其他真核生物和古细菌中已经发现了Spo11同源物，从而鉴定了一个与DNA拓扑异构酶ii相关的新蛋白家族。最后，使用亲和纯化的小鼠蛋白抗体，我们将Spo11作为单个病灶可视化，早在瘦素期，即减数分裂I中产生dsb的阶段，以及后来在精母细胞减数分裂前期的zygotene和粗线期。在粗线中，Spo11只存在于染色体完全突触的区域。令人惊讶的是，Spo11同源物在秀丽隐杆线虫和黑腹线虫的突触中是不需要的，但在减数分裂重组中却是必需的。我们已经产生了SPO11小鼠敲除，以研究该基因在哺乳动物中的生物学功能。破坏小鼠SPO11导致不孕。精母细胞由于同源突触很少或没有同源突触而在粗成期发生阻滞并发生凋亡。我们在减数分裂染色体扩散中未检测到Rad51/Dmc1病灶，表明dsb未形成。顺铂诱导的DSBs恢复了Rad51/Dmc1病灶并促进了突触。我们推测，在产生dsb后，Spo11在突触中还有一个额外的作用。最近，我们一直在进行DNA微阵列实验，以确定那些表达被DSB修饰的减数表达基因。我们一直在比较野生型、Spo11敲除型和辐照型小鼠睾丸的RNA种群。在年轻的小鼠中，由于基因敲除导致细胞凋亡，在退行性变化开始之前，只有几十个基因在Spo11 -/-中与野生型相比有差异表达。受影响最大的基因是Hop2和Mnd1基因。这些是影响酵母中同源配对的基因的同源物。我们已经在老鼠体内产生了Hop2基因的敲除。它的减数分裂表型显示一个深刻的减数分裂阻滞，这是不同于任何以前看到的。与大多数具有减数分裂表型的基因敲除不同，这些小鼠没有任何类型的突触。也就是说，尽管大多数基因敲除都显示出一些非同源突触，但来自这些小鼠的精细胞几乎没有任何突触。染色体在一定程度上是紧致的，看起来Rad51和Dmc1都正常地装饰着，就好像它们处于突触的尖端，但不能向前推进。我们现在正在检查Hop2蛋白的生化特性，以确定它们如何有助于解释这种非常不寻常的表型。

Summary of work: In order to dissect the biochemical steps involved in genetic recombination we have chosen to focus on a key early step(s): homologous pairing and strand exchange between homologous parental DNAs. A fundamental problem in homologous recombination is how the search for homology between the two DNAs is carried out. In all current models a homologous recombination protein, such as the prototypical E. coli RecA protein, loads onto a single-strand DNA generated from one duplex DNA and scans another duplex to form a synaptic (pairing) complex. Eventually, DNA strands are exchanged and a new heteroduplex is formed. While homologous pairing and strand exchange are the earliest contacts between two parental DNAs mediated by RecA and its eukaryotic homologues, Rad51 and Dmc1, homologous recombination is initiated at DNA double-strand breaks (DSBs). The protein that catalyzes DSB formation in meiosis in the budding yeast, Saccharomyces cerevisae, is the product of the SPO11 gene. Disruption of this gene results in meiotic arrest, spore lethality and a lack of meiotic recombination. Spo11 homologues have been identified in other eukaryotes and archaebacteria resulting in the identification of a new family of proteins related to DNA topoisomerase IIs. Finally, using affinity-purified antibodies to the mouse protein we have visualized Spo11 as individual foci as early as leptotene, the stage in meiosis I when DSBs are generated, and later in zygotene and pachytene of the meiotic prophase in spermatocytes. In pachytene Spo11 is found only in those areas where the chromosomes are fully synapsed. Surprisingly, Spo11 homologues are dispensable for synapsis in C. elegans and D. melanogaster yet required for meiotic recombination. We have generated a SPO11 mouse knock-out to investigate the biological function of this gene in mammals. Disruption of mouse SPO11 results in infertility. Spermatocytes arrest prior to pachytene with little or no homologous synapsis and undergo apoptosis. We did not detect Rad51/Dmc1 foci in meiotic chromosome spreads, indicating DSBs are not formed. Cisplatin-induced DSBs restored Rad51/Dmc1 foci and promoted synapsis. We speculate that there is an additional role for Spo11, after it generates DSBs, in synapsis. Recently, we have been conducting DNA microarray experiments to determine those meiotically expressed genes whose expression is modified by a DSB. We have been comparing RNA populations from testis harvested from wild type, Spo11 knock-out and irradiated mice of both types. In young mice, before degenerative changes have set in as a result of the apoptosis seen in the knockouts, there are only a few dozen genes that are differentially expressed in Spo11 -/- compared to wild type. Among the genes most affected are the Hop2 and Mnd1 genes. These are homologues of genes that affect homologous pairing in yeast. We have generated a knockout of the Hop2 gene in the mouse. Its meiotic phenotype shows a profound meiotic arrest that is unlike any seen previously. Unlike most knockouts with a meiotic phenotype these mice show no synapsis of any kind. That is, whereas most knockouts, show some willy-nilly non-homologous synapsis, spermatocytes from these mice are arrested without almost any synapsis. The chromosomes are somewhat compacted and appear normally decorated with both Rad51 and Dmc1, as if they are on the cusp of synapsis but fail to proceed forward. We are now in the process of examining the biochemical properties of the Hop2 protein to determine how they may help explain this very unusual phenotype. In all organisms, homologous recombination is inextricably related to DNA repair and replication, hence cell proliferation and its control. For example, in E. coli, RecA, the prototypical homologous recombination protein, is directly responsible for turning on the SOS response to genotoxic damage. The RecA-ssDNA- ATP filament, the active form of RecA, acts as a co-protease in the auto-catalytic digestion of the LexA repressor. Much less is known about how the SOS response is extinguished. DinI is the product of a damage-inducible, LexA-controlled gene. Previous work has shown that when this gene is over-expressed in mitomycin C-treated cells it prevent the cleavage of LexA. Recently we (in collaboration with Ben Ramirez and Ad Bax of LCP) reported a model for the abrogation of the SOS response by this SOS proein and proposed that a negatively charged helix on the C-terminus of DinI mimics DNA in its interaction with RecA, effectively short-circuiting the SOS response. Such a DNA mimic acts as a competitor for DNA on RecA. We have now identified several other proteins that have such domains that resemble this DNA mimic. We are now investigating whether these proteins bind to DNA-binding proteins in the same manner that DinI binds to RecA. Many of these proteins are of eukaryotic origin. Finally, we have used whole-genome cDNA arrays were used to analyze changes in the levels of gene expression of all E. coli ORFs after treatment with mitomycin C (MMC). Several experiments, which differ in the mode of MMC treatment, were performed, and expression profiles of E. coli cells at different time points after the addition of the DNA damaging agent were analyzed. As a whole, these experiments consist of 16 different hybridizations corresponding to about 70,000 individual data points. Around 5-10% of all genes show significant changes in their level of expression. As shown before, the expression level of several LexA-regulated genes was increased after DNA damage. On the other hand, most of those genes that show significant changes in their level of expression have not been shown previously to be inducible or repressed in the process of DNA repair. An attempt was made to classify all genes based on their responses to DNA damage. Using cluster analysis of the gene expression data it is possible to divide all the genes into at least 12 different clusters. Of the 400 or so upregulated genes about 100 were poorly annotated or not annotated at all. Of these 100 we have selected about 50 that encode for proteins of modest size, are not clearly membrane proteins and show some evolutionary conservation. We have made gene deletion strains for most of these genes and are now studying their phenotypes, both with regard to DNA metabolism (in collaboration with Sue Lovett at Brandeis) and general intermediate metabolism using the Biolog phenotypic arrays. In addition, we have initiated a structural genomics project (in collaboration with Galya Obmolova, Alex Teplyakov and Gary Gilliland at CARB) to determine the structure of as many as possible of the protein products of these 50 genes.