Molecular mimicry in the loading of a bacterial recombinase by a phage mediator

我们细胞中的遗传物质经常受到其完整性的威胁，特别是在复制以产生新的细胞副本时。多种修复和恢复途径已经发展，以确保信息从一代到一代的忠实传递。这些过程中的缺陷会导致高等生物的癌症，并严重损害细菌等简单生命形式的生存。我们小组研究重组修复途径，它利用DNA双螺旋中储存的独特核苷酸模式作为修复受损伴侣染色体的模板。在所有生物体中，相同的酶（称为RecA或Rad51）用于将单链从一个DNA螺旋交换到另一个DNA螺旋，作为损伤修复的第一步。细菌RecA和人类Rad51链交换重组酶都在DNA单链上聚合形成长的类似于DNA的结构。不幸的是，这些重组酶的装载受到单链DNA结合蛋白的阻碍，单链DNA结合蛋白保护模板免受进一步的损伤。为了克服这种潜在的严重困境，细胞含有专门的辅助蛋白，帮助RecA和Rad51进入DNA链。尽管进行了大量的研究工作，RecA和Rad51促进组装的详细机制仍不清楚。我们发现了一种新的辅助活性，来自感染细菌的病毒，具有劫持细菌RecA蛋白的能力，以促进其自身遗传物质的修复。这种蛋白质，Orf，与BRCA2有一些有趣的共同特征，有助于将Rad51重组酶加载到DNA上。BRCA2基因突变会增加患乳腺癌和卵巢癌的可能性，因为修复DNA损伤的能力降低。这项研究的目的是确定Orf的关键部分，帮助RecA克服单链DNA结合蛋白造成的障碍。我们将采用生化测定来研究Orf的存在如何影响RecA聚合物的形成。此外，我们将研究如何通过电子显微镜观察蛋白质来增强或破坏RecA细丝组装。最后，我们将研究作为双亚基环存在的Orf蛋白如何像夹子一样打开以结合单链DNA。这些结果将为遗传重组的分子机制以及这种修复过程在避免癌症发作方面的贡献提供新的见解。

The genetic material in our cells is subject to frequent threats to its integrity, especially while undergoing replication to produce new cell copies. Multiple repair and restoration pathways have evolved to ensure faithful transfer of information from generation to generation. Defects in these processes lead to cancer in higher organisms and significantly impair survival in simpler forms of life such as bacteria. Our group studies the recombinational repair pathway, which utilises the unique pattern of nucleotides stored in the DNA double helix as a template for repair of a damaged partner chromosome. In all organisms, the same enzyme (called RecA or Rad51) is utilised to exchange a single strand from one DNA helix to another as the first step in damage restoration. Both bacterial RecA and human Rad51 strand exchange recombinases polymerise on single-strands of DNA to form long filament-like structures. Unfortunately loading of these recombination enzymes is hindered by single-stranded DNA binding proteins that protect the template from further damage. To overcome this potentially serious predicament, cells contain specialized helper proteins that assist RecA and Rad51 in gaining access to the DNA strands. Despite considerable research effort, the detailed mechanism of RecA and Rad51 facilitated assembly remains unclear. We have discovered a new helper activity, from a virus infecting bacteria, with the capacity to hijack the bacterial RecA protein to promote repair of its own genetic material. This protein, Orf, has some interesting features in common with BRCA2, which helps load the Rad51 recombinase onto DNA. Mutations in the BRCA2 gene increase the likelihood of developing breast and ovarian cancers because of a reduced capacity to repair DNA damage. The research proposed in this study aims to identify the critical parts of Orf responsible for helping RecA overcome the obstruction posed by single-stranded DNA binding proteins. We will employ biochemical assays to investigate how RecA polymer formation is influenced by the presence of Orf. In addition, we will study how RecA filament assembly is enhanced or disrupted in different contexts by visualising the proteins by electron microscopy. Finally, we will investigate how the Orf protein, which exists as a dual subunit ring, can open up like a clamp to bind single-stranded DNA. The results will give fresh insight into the molecular mechanisms of genetic recombination and the contribution this repair process makes in evading the onset of cancer.