Initiation of homologous recombination by Red beta and other single strand annealing proteins

Red beta 和其他单链退火蛋白引发同源重组

• 批准号: 308554463
• 负责人: Professor Dr. Adrian Francis Stewart
• 金额: --
• 依托单位: Biotechnologisches Zentrum (Biotec)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/308554463?language=en
• 关键词: Initiation; homologous; recombination; Red; beta

Single strand annealing proteins (SSAPs) promote homologous recombination and repair of double strand breaks. The best understood SSAP, RAD52, plays a central role in the maintenance of genome integrity in eukaryotes. A prominent prokaryotic SSAP, Red beta from lambda phage, is employed in the DNA engineering technology termed recombineering. Using bioinformatics tools and biochemical evaluations, in 2009 we published that these formerly disparate proteins were ancestrally related, thereby defining an SSAP superfamily and raising the prospect that SSAPs share a unifying mechanism. Despite the importance of RAD52, Red beta and other SSAPs, there is currently no proven mechanism for DNA annealing and the initiation of homologous recombination by any SSAP. Our recent publication on Red beta (Ander et al., 2015), which will be extended in the proposed research, includes a monomer-driven DNA clamping mechanism to initiate DNA annealing that stands in stark contrast to existing models based on initiation by the ~11mer protein rings formed by RAD52 and Red beta in vitro. In the proposed research, we aim to deepen the comparison between RAD52 and Red beta by (i) obtaining Red beta structures for comparison with the published RAD52 structure and (ii) pursue biochemical and single molecule comparisons of human RAD52, Lactococcal SakRad52 and Red beta. Additionally we aim to extend our understanding of SSAP-initiated homologous recombination by further defining the mechanism of Red recombination. Previously we showed that the extraordinary efficiency of Red recombination with double stranded DNA, which underlies its remarkable usefulness in recombineering, is due to single strand annealing onto the lagging strand template at the replication fork (Maresca et al., 2010). Red recombination with double stranded DNA also depends upon the protein-protein interaction between Red beta and its partner 5 to 3 exonuclease, Red alpha. We aim to (i) identify this interaction in detail and (ii) understand how the interaction contributes to recombination.

单链退火蛋白（SSAPs）促进同源重组和修复双链断裂。最好理解的SSAP，RAD 52，在真核生物基因组完整性的维护中起着核心作用。一种突出的原核SSAP，来自λ噬菌体的Red β，用于称为重组工程的DNA工程技术中。利用生物信息学工具和生物化学评估，我们在2009年发表了这些以前不同的蛋白质是祖先相关的，从而定义了SSAP超家族，并提出了SSAP共享统一机制的前景。尽管RAD 52、Red beta和其他SSAP很重要，但目前还没有任何SSAP的DNA退火和同源重组起始机制得到证实。我们最近关于Red beta的出版物（Ander等人，2015），这将在拟议的研究中扩展，包括一个单体驱动的DNA夹紧机制，以启动DNA退火，这与现有的模型形成鲜明对比，该模型基于由RAD 52和Red beta在体外形成的~ 11聚体蛋白质环的启动。在拟议的研究中，我们的目标是通过以下方式加深RAD 52和Red beta之间的比较：（i）获得Red beta结构，用于与已发表的RAD 52结构进行比较;（ii）对人RAD 52、乳球菌SakRad 52和Red beta进行生物化学和单分子比较。此外，我们的目标是通过进一步定义Red重组的机制来扩展我们对SSAP-initiated同源重组的理解。先前，我们表明Red与双链DNA的重组的非凡效率（这是其在重组工程中的显著有用性的基础）是由于单链退火到复制叉处的滞后链模板上（Maresca等人，2010年）。Red与双链DNA的重组还取决于Red β与其配偶体5至3核酸外切酶Red α之间的蛋白质-蛋白质相互作用。我们的目标是（i）详细识别这种相互作用，（ii）了解相互作用如何有助于重组。