The role of MRNIP in replication fork stabilisation and DSB repair

基因组不稳定性，例如：高突变率和/或染色体畸变的存在-可导致癌症，促进疾病进展，并驱动遗传变异，从而导致治疗耐药性。由于基因组DNA不断受到各种DNA损伤来源的威胁，细胞已经进化出优雅的修复机制，专门处理遇到的每种类型的病变。最危险的DNA损伤是双链断裂（DSB）-这些损伤可以通过不准确的重新连接或高保真同源重组（HR）修复，这是一个使用完整的姐妹DNA序列作为模板复制回正确的DNA代码的过程。HR修复DSB的关键因素之一是肿瘤抑制因子BRCA 1/2 -在乳腺癌、卵巢癌和前列腺癌中发现突变。事实上，癌症特异性DNA修复缺陷也为实施精准医疗策略提供了机会，最近PARP抑制剂在治疗BRCA缺陷癌症方面的成功尤其证明了这一点。所有细胞在分裂之前都必须复制其DNA，而在DNA复制期间，基因组特别脆弱。DNA复制是由一个复杂的分子机器进行的，它将两条DNA链解开，形成类似于部分完成的拉链的叉状结构。许多化疗药物通过改变DNA结构并导致拉链卡住来发挥作用。如果拉链可以松开并拉上，那么电池就可以正常运行-但如果不能，拉链很容易断裂。在细胞方面，这意味着分叉崩溃，DNA受损和细胞死亡。对化疗的总体反应取决于癌细胞应对这种情况的能力。很明显，复制叉在遇到复制压力或其他遗传毒性损伤时会经历物理逆转的过程，新产生的DNA重组成一种被称为“鸡爪样”的四向结构。最近的研究表明，包括BRCA 2，BRCA 1和RAD 51在内的几种HR蛋白在促进反向叉的稳定性方面起着关键作用。BRCA 2通过将RAD 51加载到反向臂上来稳定叉-现在很明显，RAD 51加载防止了由核酸酶MRE 11和EXO 1对叉DNA的异常“咀嚼”引起的基因组不稳定性。BRCA 2的这种功能与其在HR修复DSB中的已知作用无关。尽管在理解反向分叉的生物学方面取得了重大进展，我们对MRE 11核酸酶活性如何在反向分叉处调节以防止基因组不稳定性知之甚少。我们鉴定了一种未表征的蛋白质，称为MRNIP（MRE 11-RAD 50-NBS 1-Interacting Protein）作为一种新的促进HR修复DSB的因子。我们正在进行的研究表明，MRNIP结合并稳定复制叉，促进叉进展，基因组稳定性，以及对多种化疗的抵抗力MRNIP的丢失导致显著的MRE 11依赖性复制叉降解-总体而言，我们的数据指向一种新的重要基因组稳定性机制。酶PARP 1将MRE 11招募到复制叉中-我们的初步数据表明PARP也可能招募MRNIP -事实上，MRE 11的调节剂被PARP共同招募以防止异常降解是合乎逻辑的。我们的目标是阐明MRNIP促进分叉稳定性的机制，以及DSB对电离辐射的反应，分析MRNIP结构，并开发检测MRNIP如何影响MRE 11对DNA的活性的方法。我们还将评估癌组织中的MRNIP水平- MRNIP在某些癌症中表达不足，包括卵巢腺癌（11%的癌症），因此对MRNIP水平的深入研究可以产生基于生物标志物的治疗策略或预后指示的信息。如果我们确定了MRNIP缺陷型癌症的一个子集，我们将筛选MRNIP缺陷型细胞存活所需的基因，从而确定潜在的新靶点进行治疗干预。

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.