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。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。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。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。这一发现表明，Hop2蛋白可能在使减数分裂染色体聚集在一起的过程中发挥了迄今未被认识到的核心作用。在这一生物学假说的启发下，我们纯化并研究了25kda Hop2蛋白的生化特性。最令人欣慰的是，我们已经能够证明Hop2蛋白能够促进真正同源重组蛋白特征的链入侵（d环形成）和链交换反应。值得注意的是，就非常重要的起始链入侵活性而言，它比任何一种RecA同源物都更活跃；Dmc1介导链入侵较差，而Rad51仅在Rad54蛋白存在时介导链入侵。最后，我们已经能够证明Hop2可以作为Rad51进行链交换的辅助蛋白。因此，Hop2不仅是一种真正的新型重组酶，而且可能是减数分裂HR中启动同源间接触的关键和中心蛋白。酵母中MND1基因的缺失导致了与hop2突变体非常相似的表型。此外，这两种蛋白可以从酵母减数分裂提取物中免疫沉淀。我们已经能够证实这两种蛋白可以从小鼠睾丸提取物中共免疫沉淀，此外，纯化的小鼠Hop2和Mnd1蛋白在体外相互作用并形成稳定的异源二聚体。最近，我们已经证明，这种异源二聚体，而不是单个蛋白质本身，极大地刺激了Rad51-和dmc1促进的链入侵，在后者的情况下，刺激了35倍，因此几乎70%的超螺旋底物转化为d环。此外，我们可以用纯化蛋白证明Rad51和Dmc1都与Hop2/Mnd1异源二聚体相互作用。因此，Mnd1似乎可以作为伴侣，确保Hop2配对活性仅在真核reca同源重组酶Rad51和Dmc1存在时才被激发。我们已经能够制备突变的Hop2和Mnd1蛋白，这使我们能够解剖这些蛋白刺激Dmc1链入侵活性的机制。结果表明，Hop2/Mnd1异源二聚体刺激Dmc1既不需要Hop2的内在链入侵活性，也不需要Hop2/Mnd1的DNA结合活性。此外，Hop2/Mnd1异源二聚体刺激Dmc1与单链DNA结合30倍。此外，哺乳动物Hop2/Mnd1复合体促进了ssDNA-Dmc1核蛋白丝捕获dsDNA。此外，异二聚体还能稳定DNA-Dmc1细丝。最后，Mnd1基因敲除小鼠的表型强烈表明，正如在酵母中所显示的那样，小鼠dsb减数分裂修复至少有两条主要途径，而Hop2在这两条途径中都起着重要作用。

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. 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. Recently, we have been conducting DNA microarray experiments to determine those meiotically expressed genes whose expression is modified by a DSB. 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. This finding suggests that the Hop2 protein might play a heretofore-unrecognized central role in bringing meiotic chromosomes together. Prompted by this biologically inspired hypothesis we have purified and studied the biochemical properties of the 25 Kda Hop2 protein. Most gratifyingly, we have been able to show that Hop2 protein can promote the strand invasion (D-loop formation) and strand exchange reactions characteristic of bona fide homologous recombination proteins. Remarkably, with respect to the very important initiating strand invasion activity it is more active than either RecA homologue; Dmc1, mediates strand invasion poorly and Rad51, mediates strand invasion only in the presence of the Rad54 protein. Finally, we have been able to show that Hop2 can act as an accessory protein for the strand exchange carried out by Rad51. Thus, Hop2 is not only a true novel recombinase but also may be the crucial and central protein in initiating interhomolog contacts in meiotic HR. Deletion of the MND1 gene in yeast results in a very similar phenotype to that observed in a hop2 mutant. Furthermore, the two proteins could be immunoprecipitated from yeast meiotic extracts. We have been able to confirm that the two proteins could be coimmunoprecipitated from mouse testes extracts and that, in addition, purified mouse Hop2 and Mnd1 proteins interact in vitro and form a stable heterodimer. Recently, we have shown that this heterodimer, but not the individual proteins by themselves, greatly ssstimulates Rad51- and Dmc1-promoted strand invasion, in the latter case by 35-fold so that almost 70% of the supercoiled substrate is converted to D-loops. Furthermore, we can demonstrate with purified proteins that both Rad51 and Dmc1 interact with the Hop2/Mnd1 heterodimer. Thus, it appears that Mnd1 may act as a chaperone that ensures that the Hop2 pairing activity is only elicited in the presence of the eukaryotic RecA-homologous recombinases, Rad51 and Dmc1. We have been able to prepare mutant Hop2 and Mnd1 proteins that have enabled us to dissect the mechanism by which these proteins stimulate the strand invasion activity of Dmc1. The results indicate that neither the intrinsic strand invasion activity of Hop2 nor the Hop2/Mnd1 DNA binding activity is required for the stimulation of Dmc1 by the Hop2/Mnd1 heterodimer. Furthermore, the Hop2/Mnd1 heterodimer stimulates the binding of Dmc1 to single-strand DNA by 30-fold. Furthermore, the mammalian Hop2/Mnd1 complex facilitates the capture of dsDNA by a ssDNA-Dmc1 nucleoprotein filament. In addition, the heterodimer stabilizes DNA-Dmc1 filaments. Finally, the phenotype of an Mnd1 knockout mouse strongly suggests that, as has been shown in yeast, there are at least two major pathways for the meiotic repair of DSBs in mice and that Hop2 is an essential player in both of these pathways. In the past few years it has become increasingly clear that recombination events are distributed non-randomly in the genomes of eukaryotes. We have used recent developments in computational methods and the availability of extensive human genetic variation data from the HapMap project to generate a high-resolution map of historically stable recombination hotspots of the human genome. Analysis of the recombination rate map revealed the presence of numerous well-defined hotspots of varying strength. We have found a good correlation between the hotspots predicted by us using computational methods and the hotspots previously identified by the analysis of recombination breakpoints in well-studied genomic regions. The overall distribution of hotspots is non-random. There is a correlation of the distribution of hotspots with various genomic and chromosomal features.