The role of MRNIP in replication fork stabilisation and DSB repair

MRNIP 在复制叉稳定和 DSB 修复中的作用

• 批准号: MR/S034579/1
• 负责人: Christopher Staples
• 金额: $ 180.45万
• 依托单位: Bangor University
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FS034579%2F1
• 关键词: role; MRNIP; replication; fork; stabilisation

Genome instability eg: a high mutation rate and/or the presence of chromosomal aberrations - can cause cancer, promote disease progression and drive the genetic variation underpinning therapeutic resistance. Since genomic DNA is constantly under threat from various sources of DNA damage, the cell has evolved elegant repair mechanisms to deal specifically with each type of lesion encountered. The most dangerous DNA lesions are Double-Strand Breaks (DSBs) - these are repaired either by inaccurate rejoining or by high-fidelity Homologous Recombination (HR), a process that uses the intact sister DNA sequence as a template to copy back the correct DNA code. Among the crucial players in DSB repair by HR are the tumour suppressors BRCA1/2 - found mutated in breast, ovarian and prostate cancers. Indeed, cancer-specific DNA repair defects also provide opportunities to implement precision medicine strategies, notably evidenced by the recent success of PARP inhibitors in the treatment of BRCA-deficient cancers.All cells must copy their DNA before they can divide and during DNA replication the genome is particularly vulnerable. DNA replication is carried out by a complex molecular machine that unwinds the two DNA strands, forming a fork-like structure akin to a partially done zipper. Many chemotherapy drugs work by altering DNA structure and causing the zipper to get stuck. If the zipper can be freed and zipped up, then the cell can proceed normally - but if it cannot, the zipper is prone to breakage. In cellular terms, this entails fork collapse, damaged DNA and cell death. The overall response to chemotherapy is determined by the ability of the cancer cell to deal with this scenario. It has become apparent that replication forks undergo a process of physical reversal upon encountering replication stresses or other genotoxic insults, the newly-made DNA reforms into a four-way structure described as 'chicken foot-like'. Recent work demonstrates crucial roles for several HR proteins including BRCA2, BRCA1, and RAD51 in promoting the stability of reversed forks. BRCA2 stabilises the fork by loading RAD51 onto the reversed arm - it is now apparent that RAD51 loading prevents genome instability induced by aberrant 'chewing' of the fork DNA by the nucleases MRE11 and EXO1. This function of BRCA2 is independent of its known role in DSB repair by HR. Despite significant advances in understanding the biology of the reversed fork, how MRE11 nuclease activity is regulated at reversed forks to prevent genome instability is poorly understood.We identified an uncharacterised protein called MRNIP (MRE11-RAD50-NBS1-Interacting Protein) as a novel factor that promotes DSB repair by HR. Our ongoing studies show that MRNIP binds to and stabilises replication forks, promoting fork progression, genome stability, and resistance to multiple chemotherapies. Loss of MRNIP results in marked MRE11-dependent replication fork degradation - overall our data points to a novel important genome stability mechanism. The enzyme PARP1 recruits MRE11 to replication forks - our initial data suggests that PARP may also recruit MRNIP - indeed, it is logical that regulators of MRE11 are co-recruited by PARP to prevent aberrant degradation. Our goals are to elucidate the mechanisms via which MRNIP promotes fork stability, as well as the DSB response to ionising radiation, to analyse MRNIP structure, and to develop assays to test how MRNIP influences MRE11 activity against DNA.We will also assess MRNIP levels in cancer tissues - MRNIP is underexpressed in some cancers including ovarian adenocarcinoma (11% of cancers), and thus an in-depth study of MRNIP levels could yield information leading to biomarker-based treatment strategies or prognostic indication. Should we identify a subset of MRNIP-deficient cancers, we will screen for genes that are required for survival of MRNIP-deficient cells, thus identifying potential novel targets for therapeutic intervention.

基因组不稳定性，例如：高突变率和/或染色体畸变的存在 - 会导致癌症，促进疾病进展并驱动遗传变异，而遗传变异是治疗性抗性的基础。由于基因组DNA不断受到DNA损伤各种来源的威胁，因此该细胞发展出优雅的修复机制，以专门处理遇到的每种类型的病变。最危险的DNA病变是双链断裂（DSB） - 通过不准确的重新加入或高保真同源重组（HR）来修复这些损伤，该过程将完整的姐妹DNA序列用作模板以复制正确的DNA代码。在DSB修复中，HR的关键参与者中有肿瘤抑制剂BRCA1/2-发现在乳腺，卵巢和前列腺癌中突变。实际上，癌症特异性的DNA修复缺陷还提供了实施精确医学策略的机会，特别是由PARP抑制剂在治疗BRCA缺陷癌症治疗的最新成功中所证明的。所有细胞必须在它们可以分裂之前复制其DNA，并且在DNA复制过程中，基因组特别容易受到攻击。 DNA复制是通过一个复杂的分子机进行的，该机器将两个DNA链放开，形成类似于部分完成拉链的叉状结构。许多化学疗法药物通过改变DNA结构并导致拉链被卡住来起作用。如果可以释放拉链并拉紧拉链，则可以正常进行单元格 - 但是如果不能进行，则拉链很容易断裂。用细胞术语来说，这需要叉子崩溃，DNA和细胞死亡受损。对化学疗法的总体反应取决于癌细胞处理这种情况的能力。显然，复制叉会在遇到复制应力或其他遗传毒性损伤后经历物理逆转的过程，将新制作的DNA改革变成了一种被描述为“类似鸡足样的”的四向结构。最近的工作表明，多种HR蛋白（包括BRCA2，BRCA1和RAD51）在促进反向分叉的稳定性中的关键作用。 BRCA2通过将RAD51加载到反向的臂上来稳定叉子 - 现在显然，RAD51载荷可以防止因核酸酶MRE11和EXO1的异常“咀嚼”叉子DNA引起的基因组不稳定性。 BRCA2的这一功能与HR在DSB修复中的已知作用无关。尽管在理解反向叉的生物学方面取得了重大进展，但如何在反向叉子上调节MRE11核酸酶活性以预防基因组稳定性。我们正在进行的研究表明，mRNIP与复制叉结合并稳定，促进分叉进展，基因组稳定性以及对多种化学疗法的耐药性。 MRNIP的丢失导致MRE11依赖性复制叉降解 - 总体而言，我们的数据指向了一种新型重要的基因组稳定机制。酶PARP1募集MRE11复制叉 - 我们的初始数据表明PARP也可以募集MRNIP-实际上，MRE11的调节剂由PARP共同授予以防止异常降解。 Our goals are to elucidate the mechanisms via which MRNIP promotes fork stability, as well as the DSB response to ionising radiation, to analyse MRNIP structure, and to develop assays to test how MRNIP influences MRE11 activity against DNA.We will also assess MRNIP levels in cancer tissues - MRNIP is underexpressed in some cancers including ovarian adenocarcinoma (11% of cancers), and thus an对MRNIP水平的深入研究可能会产生信息，从而导致基于生物标记的治疗策略或预后指示。如果我们确定缺乏mRNIP的癌症的子集，我们将筛选出MRNIP缺陷细胞存活所需的基因，从而确定潜在的治疗干预靶标。

项目成果

Assessment of DNA fibers to track replication dynamics.

评估 DNA 纤维以跟踪复制动态。

• DOI: 10.1016/bs.mcb.2023.02.007
• 发表时间: 2024
• 期刊: Methods in cell biology
• 作者: Bennett LG