Alcon-et-al-2024-FANCD2I

Raw data generated for the purpose of the research project published under the title: "FANCD2-FANCI surveys DNA and recognises double to single-stranded junctions" ARTICLE ABSTRACT: DNA crosslinks block DNA replication and are repaired by the Fanconi Anemia pathway. The FANCD2-FANCI (D2-I) protein complex is central to this process as it initiates repair by coordinating DNA incisions around the lesion 1. However, D2-I is also known to play a more general role in DNA repair and in protecting stalled replication forks from unscheduled degradation 2-4. It is currently unclear how DNA crosslinks are recognized and how D2-I functions in replication fork protection. Here, using single-molecule imaging, we show that D2-I is a sliding clamp that binds to and diffuses on double-stranded DNA. Strikingly, sliding D2-I stalls upon encountering single-stranded–double-stranded (ss-ds) DNA junctions, structures that are generated when replication forks stall at DNA lesions 5. Using cryoEM, we determined structures of D2-I on DNA which show that stalled D2-I makes specific interactions with the ss-dsDNA junction that are distinct from those made by sliding D2-I. Thus, D2-I surveys dsDNA and, when it reaches a ssDNA gap, it specifically clamps onto ss-dsDNA junctions. Since ss-dsDNA junctions are found at stalled replication forks, D2-I can identify sites of DNA damage. Therefore, our data provide a unified molecular mechanism that reconciles the roles of D2-I in recognition and protection of stalled replication forks in multiple DNA repair pathways.本研究项目生成的原始数据，相关研究成果发表于题为《FANCD2-FANCI扫描DNA并识别双链-单链连接位点》的学术论文。 论文摘要： DNA交联会阻断DNA复制，并通过范可尼贫血（Fanconi Anemia）通路完成修复。FANCD2-FANCI（D2-I）蛋白复合物是该通路的核心组分，它通过协调损伤位点周围的DNA切口启动修复过程¹。现有研究表明，D2-I还在DNA修复以及保护停滞的复制叉免受异常降解中发挥更为广泛的作用²⁻⁴。目前学界尚未明确DNA交联的识别机制，以及D2-I在复制叉保护中的具体功能机制。本研究借助单分子成像（single-molecule imaging）技术，证实D2-I是一类可结合双链DNA（double-stranded DNA, dsDNA）并在其上滑动扩散的滑动夹（sliding clamp）。值得注意的是，滑动状态的D2-I在遭遇单链-双链（single-stranded–double-stranded, ss-ds）DNA连接点时会发生停滞——这类结构是复制叉在DNA损伤处停滞时所产生的⁵。我们通过冷冻电镜（cryoEM）解析了D2-I与DNA结合的复合物结构，结果显示停滞状态的D2-I与ss-dsDNA连接点存在特异性相互作用，该相互作用模式与滑动状态下D2-I的结合方式截然不同。综上，D2-I可扫描双链DNA，当其抵达单链DNA缺口时，会特异性地钳合在ss-dsDNA连接位点上。由于停滞的复制叉处普遍存在ss-dsDNA连接点，因此D2-I能够精准识别DNA损伤位点。本研究的数据为统一的分子机制提供了实验依据，该机制可阐释D2-I在多种DNA修复通路中，识别并保护停滞复制叉的双重功能。