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DNA strand break repair and links to human disease

Periodic Reporting for period 3 - DNA2REPAIR (DNA strand break repair and links to human disease)

Reporting period: 2018-09-01 to 2020-02-29

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.
Good progress has been made in the first 3-4 years of the project, as follows:

Specific aim 1: Mechanistic and structural analyses of pre-recombination complexes
DNA strand breaks are repaired by a process called homologous recombination repair. This requires a number of cellular factors including the BRCA2 and PALB2 tumour suppressor proteins, RAD51, RAD52, RAD54, the five RAD51 parlous (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3) and RPA. All proteins have been purified and we are carrying biochemical and structural analyses of the proteins in order to determine the precise roles that they play in DNA repair. Our work revealed that RAD52 forms two distinct structures. One is an eleven subunit ring, the structure of which has been solved at high resolution, and the second is an open ring structure. We find that the open ring interacts with RPA and DNA, and that this is the functional form of the protein in DNA repair. we are currently analysing the coordinated actions of BRCA2-PALB2, RAD52, RAD51 and RPA in assembling pre-recombination complexes in vitro, and will develop even more complete biochemical reactions to determine their mode of action.
In the clinic, breast and ovarian cancer patients with BRCA germline mutations show a good response rate to treatment with a PARP inhibitor (Olaparib) and PARPi are now used as a first-line therapy against certain ovarian cancers. Unfortunately, after 3-4 years, cancer cells develop resistance to PARPi treatment. We therefore sought to find new mechanism by which the potency of PARPi therapy could be increased. We found that inactivation of a nucleotide salvage pathway protein significantly sensitised BRCA cells to the PARPi, and are moving forward in studies to discover drugs that can inhibit this pathway. We believe it may provide a promising strategy for more efficient killing of BRCA-deficient cancers.

Specific Aim 2: Structure-Selective Endonucleases
For several years, we have been interested in understanding the roles of structure-selective endonucleases such as SLX1-SLX4, MUS81-EME1 and GEN1 in the resolution of recombination intermediates. We discovered that SLX1-SLX4-MUS81-EME1-XPF-ERCC1 (SMX) complex forms at prometaphase, in response to CDK/PLK1 phosphorylation events and that this complex plays a key role in the resolution of DNA recombination intermediates. Mutations in key components of this complex lead to an inheritable disease known as Fanconi anaemia, and individuals with this disease are predisposed to cancer. Recently, we discovered that the MutSb mismatch repair protein interacts with recombination intermediates and with SLX4. Together MutSb and SMX are significantly more active than SMX alone, and these interactions are required for the resolution of recombination intermediates. These are remarkable observations as cell cycle-dependent formation and activation of this tri-nuclease complex appears to provide a unique mechanism by which cells ensure chromosome segregation and preserve genome integrity.

Specific Aim 3: Senataxin and the Maintenance of Genome Stability in Neuronal Cells
The neurodegenerative disorders ataxia with oculomotor apraxia-2 is caused by mutations in the SETX gene. Individuals with this autosomal recessive cerebellar ataxia exhibit motor neuron degeneration, together with progressive muscle weakness and atrophy. Typically, motor coordination is affected at an early age (2 to 6 years) and progressive disability continues leading to confinement to a wheelchair in adolescent life. We found that mutations in the gene, SETX, that causes AOA2 are sensitive to agents that cause oxidative stress (H2O2), DNA replication stress (HU, aphidicolin), or induce the formation of R-loops (Diospyrin D1). In Senataxin- depleted cells, and also in AOA2 patient-derived cells, we observed increased DNA break formation at mitosis. Over-expression of RNase H1, or treatment with the transcription chain terminator Cordycepin, reduced the frequency of these breaks indicating that R-loops were the underlying cause of genome instability after Senataxin deficiency. We have now extended these studies by combining genomic and transcriptomic approaches and found that loss of Senataxin from cells derived from AOA2 patients leads to a genome-wide increase in RNA polymerase II (RNAPII) pausing events, R-loop accumulation, altered gene expression and transcription stress-induced genomic instability across genes and at fragile sites.
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.