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ERC

DNA2REPAIR Report Summary

Project ID: 666400
Funded under: H2020-EU.1.1.

Periodic Reporting for period 1 - DNA2REPAIR (DNA strand break repair and links to human disease)

Reporting period: 2015-09-01 to 2017-02-28

Summary of the context and overall objectives of the project

Our genetic material is continually subjected to damage, either from endogenous sources such as reactive oxygen species, produced as by-products of oxidative metabolism, from the breakdown of replication forks during cell growth, or by agents in the environment such as ionising radiation or carcinogenic chemicals. To cope with DNA damage, cells employ elaborate and effective repair processes that specifically recognise a wide variety of lesions in DNA. These repair systems are essential for the maintenance of genome integrity. Unfortunately, some individuals are genetically predisposed to crippling diseases or cancers that are the direct result of mutations in genes involved in the DNA damage response. For several years our work has been at the forefront of basic biological research in the area of DNA repair, and in particular we have made significant contributions to the understanding of inheritable diseases such as breast cancer, Fanconi anemia, and the neurodegenerative disorder Ataxia with Oculomotor Apraxia (AOA). The focus of this ERC proposal is: (i) to determine the mechanism of action and high-resolution structure of the BRCA2 breast cancer tumour suppressor, and to provide a detailed picture of the interplay between BRCA2, PALB2, RAD51AP1 and the RAD51 paralogs, in terms of RAD51 filament assembly/disassembly, using biochemical, electron microscopic and cell biological approaches, (ii) to determine the biological role of a unique six-subunit structure-selective tri-nuclease complex (SLX1-SLX4-MUS81-EME1-XPF-ERCC1), with particular emphasis on its roles in DNA crosslink repair and Fanconi anemia, and (iii) to understand the actions of Senataxin, which is defective in AOA2, in protecting against genome instability in neuronal cells. These three distinct and yet inter-related areas of the research programme will provide an improved understanding of basic mechanisms of DNA repair and thereby underpin future therapeutic developments that will help individuals afflicted with these diseases.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Good progress has been made during the first 18 months, as follows:

1. MUS81-EME1 vs MUS81-EME2
To define the timely association of MUS81 with either EME1 or EME2 throughout the cell cycle, we analysed protein extracts from cells synchronized at different cell cycle stages. We found that the relative amounts of MUS81-EME1 and MUS81-EME2 remained constant throughout the cell cycle. Previously (Wyatt et al., Molecular Cell 2013), we showed that MUS81-EME1 associates with SLX4 during G2/M, to form the MUS81-EME1-SLX4-XPF-ERCC1 (SMX) complex. Within SMX, the nuclease activity of MUS81 is increased (Wyatt et al., Molecular Cell 2017). MUS81-EME2 activity on recombination intermediates also peaks at G2/M and again is dependent on SLX4. However, our studies support an essentially distinct role of MUS81-EME2 in the processing of replication fork structures.

To determine the post-translational modifications that occur, we performed mass spectometry analyses of immunopurified, FLAG-tagged MUS81, EME1 and EME2 and detected a number of putative, cell cycle-dependent phosphorylation and ubiquitylation sites on both EME1 and EME2. The biological significance of these phosphorylations and ubiquitylations to the activity of MUS81 in DNA repair, are being analysed using CRISPR KO cell lines of MUS81, EME1 and EME2.

2. Identification of a novel form of MUS81
During our studies of MUS81 we discovered the presence of a shorter isoform of MUS81, which appears to be an alternative transcript (MUS81short) starting from the downstream methionine 75. Interestingly, MUS81short lacks the N-terminal HhH domain, which is required for interaction with SLX4, and is similar to a truncated version of MUS81 that displays increased activity in vitro (Wyatt et al., Molecular Cell 2017). To explore the specific role of MUS81short-EME2, we have generated MUS81long-, MUS81short-, EME1- and EME2-deficient cell lines using CRISPR technology and we are currently assessing the contribution of these factors in genome integrity and in cell survival after treatment with DNA damaging or fork-blocking drugs.

3. Biochemical analysis of the SMX tri-nuclease complex
The SMX complex plays a key role in the removal of replication and recombination intermediates and is essential for the maintenance of genome stability. Resolution of these potentially toxic structures was found to require the MUS81-EME1 endonuclease, which is activated at prometaphase by formation of the SMX tri-nuclease containing three DNA repair structure-selective endonucleases: SLX1-SLX4, MUS81-EME1 and XPF-ERCC1. Remarkably, we found that SMX tri-nuclease is more active than the three individual nucleases, efficiently cleaving replication forks and recombination intermediates (Wyatt et al., Molecular Cell 2017). Within SMX, SLX4 co-ordinates the SLX1 and MUS81-EME1 nucleases for Holliday junction resolution, in a reaction stimulated by XPF-ERCC1. SMX formation activates MUS81-EME1 for replication fork and flap structure cleavage by relaxing substrate specificity. Activation involves MUS81’s conserved N-terminal HhH domain, which mediates incision site selection and SLX4-binding. Cell cycle-dependent formation and activation of this tri-nuclease complex provides a unique mechanism by which cells ensure chromosome segregation and preserve genome integrity.

4. Molecular analysis of cells defective for Senataxin
Senataxin (SETX), a putative RNA/DNA helicase mutated in the neurodegenerative disorders AOA2 and ALS4, is considered to be an important player in the resolution of RNA/DNA hybrids (R-loops) that are formed either during transcription cycle or upon transcription stalling events. However, the molecular mechanisms of action of SETX and its connection to neurodegeneration are not fully understood. To address the aims of the proposed study, we previously established multiple AOA2 patient-derived lymphoblastoid and fibroblast cell lines along with age- and sex-matched controls. In this 18 month period, we also successfully generated human (HAP1, HEK293) and mouse (NSC-34 motor neuron-like) SETX knockout (K/O) cells using CRISPR/Cas9 technology. In order to further validate our findings, we have also included the SETX+/+ and SETX-/- mouse embryonic fibroblasts.

Using multi-colour FISH (M-FISH) analyses, we found that SETX K/O and AOA2 cells are prone to increased frequency of chromosome abnormalities, including deletions, duplications, translocations and few numerical abnormalities. Furthermore, SETX K/O and AOA2 cells accumulate high levels of constrictions/gaps/breaks/radials on metaphase chromosomes and 53BP1 G1-nuclear bodies, upon treatment with agents that stall replication or transcription. These phenotypes are significantly reduced by overexpression of RNaseH1 or by treatment with transcription terminator (cordycepin). To identify the hotspots of genomic instability in SETX-defective cells, we performed array comparative genomic hybridization (aCGH) with DNA isolated from control- and SETX K/O or AOA2 cells, and found that the majority of chromosomal gains and losses occurred at genomic regions associated with one or more genes. Interestingly, RNA polymerase II (RNAPII) ChIP-sequencing experiments with human SETX K/O and control cells revealed that the absence of SETX caused a genome-wide increase in RNAPII stalling or pausing events (transcription stress) over transcription start sites (TSS). We therefore measured RNAPII stalling index of SETX K/O and control cells by calculating ratio of RNAPII density near TSS (-250 to +250 bp) and RNAPII density across gene bodies (+250 bp to transcription termination site), and found that ~40% of the 16,191 RNAPII genes analysed show RNAPII stalling index of K/O vs. control 2. Consistent with these findings, transcriptome studies revealed that SETX K/O and AOA2 cells exhibit altered gene expression as compared to control cells. For instance, gene expression analyses of all AOA2 and control lymphoblastoid cell lines revealed a total of 569 genes (291 upregulated and 278 downregulated) that are differentially expressed in AOA2 (FDR correcting p-value of 0.05 and absolute fold change of 1.5). Interestingly, Gene Set Enrichment analysis of microarray data revealed that DNA integrity checkpoint genes (ATR, CHK1, CHK2, NBS1) are upregulated in AOA2-patient cell lines (nominal p-value < 0.05). Taken together, our results indicate that transcription stress act as a major source of genomic instability in cells lacking SETX.

The molecular basis of the recruitment of SETX to RNAPII TSS is not clearly understood. Using large-scale immunoprecipitation followed by mass spectrometry, we identified human SETX interacts with many proteins implicated in RNA metabolism. We confirmed that SETX, RNA helicase DDX21 and the transcriptional regulator KAP1 constitutively associate with each other, and are present together at TSS. Furthermore, we found that KAP1 is necessary for the formation of the SETX-DDX21 complex at TSS. Interestingly, SETX-DDX21 complex recruits the RNA exosome component RRP6 to degrade R-loops. Collectively, our results suggest that SETX-DDX21-RRP6 (S-D-R) complex promotes R-loop repair (RLR) at sites of transcription stress and transcription-replication conflicts. In the absence of S-D-R complex, R-loop containing transcription bubbles are targeted and cleaved by transcription-coupled repair (TCR) proteins (CSB, XPG, XPF), which in turn promote the formation of chromosome fragility observed in SETX K/O cells.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The studies indicated above are at the cutting edge of the field, and increase our knowledge of the molecular basis of genome stability. Without such mechanisms of DNA manipulation and repair, cells are genetically unstable and show many of the hallmarks that lead to tumorigenesis. The studies therefore underpin our understanding of how cells respond to DNA damage and avoid tumorigenesis.
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