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Classically, p53 tumor suppressor acts in transcription, apoptosis, and cell cycle arrest. Yet, replication-mediated genomic instability is integral to oncogenesis, and p53 mutations promote tumor progression and drug-resistance. By delineating human and murine separation-of-function p53 alleles, we find that p53 null and gain-of-function (GOF) mutations exhibit defects in restart of stalled or damaged DNA replication forks that drive genomic instability, which isgenetically separable from transcription activation. By assaying protein-DNA fork interactions in single cells, we unveil a p53-MLL3-enabled recruitment of MRE11 DNA replication restart nuclease. Importantly, p53 defects or depletion unexpectedly allow mutagenic RAD52 and POLθ pathways to hijack stalled forks, which we find reflected in p53 defective breast-cancer patient COSMIC mutational signatures. These data uncover p53 as a keystone regulator of replication homeostasis within a DNA restart network. Mechanistically, this has important implications for development of resistance in cancer therapy. Combined, these results define an unexpected role for p53-mediated suppression of replication genome instability. When a cell divides to make more cells, it duplicates its DNA to pass on an identical set of genes to the new cell. Copying DNA – also known as DNA replication – is a complex process that involves several steps. First, the double helix gradually unwinds and unzips to separate the DNA strands. This creates a molecule known as the ‘replication fork’. Then, copies of each strand are created and proofread for errors. Eventually, the strands are sealed back together so that the helices contain one old and one new part. But sometimes errors sneak in during DNA replication, which can lead to mutations that may cause cancer. The higher the number of mutations, the bigger the chance is that the cancer becomes aggressive and resistant to therapy. Some of the most common mutations found in tumors happen in a protein called p53. This protein is known to stop tumors from growing by selectively killing cells with mutations. When p53 is faulty, mutant cells no longer die and can grow uncontrollably to form tumors. However, its killing abilities do not fully explain how p53 protects cells from accumulating mutations that can cause cancer, and until now, it was not known if p53 also had any other roles. Now, Schlacher et al. discovered that p53 can protect the DNA from mutations. The experiments used normal cells and cancer cells from humans and mice, in which p53 was either blocked or modified. The experiments revealed that p53 plays an important role during DNA replication. When p53 is ‘healthy’, it binds to the replication fork. This ensures that replication restarts properly after it has passed faulty patches of the DNA. The p53 protein also helps to organize the proteins involved in DNA replication. When p53 was absent or mutated, the DNA-repair protein that usually binds to the fork failed to attach properly. Instead, other proteins prone to make mutations took over the replication fork and created a pattern of mutations commonly found in tumors resistant to treatment. A next step will be to investigate p53’s role at damaged DNA replication forks and how it interacts with other proteins involved in DNA replication. To fully understand all roles that p53 plays in preventing tumor growth can help to find new ways to treat tumors with p53 defects or tumors that have become resistant to treatment.

Growth factor receptor-bound protein 2 (GRB2) is a cytoplasmic adapter for tyrosine kinase signaling and a nuclear adapter for homology-directed-DNA repair. Here we find nuclear GRB2 protects DNA at stalled replication forks from MRE11-mediated degradation in the BRCA2 replication fork protection axis. Mechanistically, GRB2 binds and inhibits RAD51 ATPase activity to stabilize RAD51 on stalled replication forks. In GRB2-depleted cells, PARP inhibitor (PARPi) treatment releases DNA fragments from stalled forks into the cytoplasm that activate the cGAS–STING pathway to trigger pro-inflammatory cytokine production. Moreover in a syngeneic mouse metastatic ovarian cancer model, GRB2 depletion in the context of PARPi treatment reduced tumor burden and enabled high survival consistent with immune suppression of cancer growth. Collective findings unveil GRB2 function and mechanism for fork protection in the BRCA2-RAD51-MRE11 axis and suggest GRB2 as a potential therapeutic target and an enabling predictive biomarker for patient selection for PARPi and immunotherapy combination. GRB2 is known for its role in Receptor Tyrosine Kinase and RAS signaling. Here the authors unveil a GRB2 function and mechanism for DNA replication fork protection. GRB2 alleviates oncogenic replication stress, and in doing so, averts cancer immune destruction by inhibiting cGAS/STING and pro-inflammatory cytokine production.

RAD51C is an enigmatic predisposition gene for breast, ovarian, and prostate cancer. Currently, missing structural and related functional understanding limits patient mutation interpretation to homology-directed repair (HDR) function analysis. Here we report the RAD51C-XRCC3 (CX3) X-ray co-crystal structure with bound ATP analog and define separable RAD51C replication stability roles informed by its three-dimensional structure, assembly, and unappreciated polymerization motif. Mapping of cancer patient mutations as a functional guide confirms ATP-binding matching RAD51 recombinase, yet highlights distinct CX3 interfaces. Analyses of CRISPR/Cas9-edited human cells with RAD51C mutations combined with single-molecule, single-cell and biophysics measurements uncover discrete CX3 regions for DNA replication fork protection, restart and reversal, accomplished by separable functions in DNA binding and implied 5’ RAD51 filament capping. Collective findings establish CX3 as a cancer-relevant replication stress response complex, show how HDR-proficient variants could contribute to tumor development, and identify regions to aid functional testing and classification of cancer mutations. In this study, the authors present structures and functional analyses for the RAD51C-XRCC3 tumor suppressor complex, providing insights into recurrent mutations in cancer and Fanconi Anemia patients that uncover distinct DNA replication fork protection, restart and reversal regions.

The prototypic cancer-predisposition disease Fanconi Anemia (FA) is identified by biallelic mutations in any one of twenty-three FANC genes. Puzzlingly, inactivation of one Fanc gene alone in mice fails to faithfully model the pleiotropic human disease without additional external stress. Here we find that FA patients frequently display FANC co-mutations. Combining exemplary homozygous hypomorphic Brca2/Fancd1 and Rad51c/Fanco mutations in mice phenocopies human FA with bone marrow failure, rapid death by cancer, cellular cancer-drug hypersensitivity and severe replication instability. These grave phenotypes contrast the unremarkable phenotypes seen in mice with single gene-function inactivation, revealing an unexpected synergism between Fanc mutations. Beyond FA, breast cancer-genome analysis confirms that polygenic FANC tumor-mutations correlate with lower survival, expanding our understanding of FANC genes beyond an epistatic FA-pathway. Collectively, the data establish a polygenic replication stress concept as a testable principle, whereby co-occurrence of a distinct second gene mutation amplifies and drives endogenous replication stress, genome instability and disease. Tomaszowski et al show that co-mutations in Brca2 and Rad51c synergistically drive cancer and developmental disease, which was unexpected given their epistatic DNA repair roles, and expands our understanding of their tumor suppressive functions.

The classical non-homologous end-joining (c-NHEJ) pathway is largely responsible for repairing DNA double-strand breaks (DSBs) in mammalian cells. Absence of c-NHEJ causes genomic instability in mice and leads to deficiencies in V(D)J recombination. XLF, (the last bona fide c-NHEJ factor discovered) is known to stimulate ligation by the core ligation complex: XRCC4-Ligase 4. However, the precise mechanism by which XLF stimulates XRCC4-Ligase 4 mediated DNA ligation is not well understood. Recent structural studies have shown that XLF can interact with XRCC4 to form filaments of alternating XRCC4 and XLF dimers; these filaments mediate DNA end bridging in vitro, providing a potential mechanism by which XLF might stimulate ligation. Here, we show that disrupting the interaction between XRCC4 and XLF by XRCC4 mutation, thereby abolishing filament formation, affects V(D)J recombination in cells and hinders the ability of cells expressing these mutants to survive in response to zeocin, a radiomimetic drug. Furthermore, we characterize an XLF mutant (L115A) that does not interact with XRCC4, and thus does not form filaments or bridge DNA in vitro. However, this mutant is fully sufficient in stimulating ligation of either blunt or cohesive DNA ends by X4/Lig4 in vitro. This separation of function mutant fully complements the zeocin sensitive phenotype and V(D)J recombination deficits of some XLF deficient cell strains but not others, suggesting a variable requirement for DNA bridging in different cell types. To determine whether lack of XRCC4/XLF bridging might be compensated for by other factors, candidate repair factors were disrupted in XLF or XRCC4 deficient cells. Loss of either ATM or the newly described XRCC4/XLF like factor, PAXX accentuates the cellular requirement for XLF. In the case of ATM/XLF loss, the increased cellular requirement can be attributed to its bridging function; however in case of PAXX/XLF loss the increased requirement for XLF is independent of bridging.

Anemia of inflammation (AI) is a common comorbidity associated with obesity, diabetes, cardiac disease, aging, and during anti-cancer therapies. Mounting evidence illustrates that males are disproportionally affected by AI, but not why. Here we demonstrate a molecular cause for a sex-bias in inflammation. The data shows that mitochondrial DNA (mtDNA) instability induced by dietary stress causes anemia associated with inflamed macrophages and improper iron recycling in mice. These phenotypes are enhanced in mice with mutations in , which predisposes to the progeroid disease Fanconi Anemia. The data reveals a striking sex-bias whereby females are protected. We find that estrogen acts as a mitochondrial antioxidant that reduces diet-induced oxidative stress, mtDNA replication instability and the distinctively mtDNA-dependent unphosphorylated STAT1 response. Consequently, treatment of male mutant mice with estrogen or mitochondrial antioxidants suppresses the inflammation-induced anemia. Collectively, this study uncovers estrogen-responsive mtDNA replication instability as a cause for sex-specific inflammatory responses and molecular driver for AI.

DNA replication stress responses are key genomic stability guardians critical during development, aging, hematopoiesis, disease suppression and cancer therapy response. Reversed forks (RF) form at stalled DNA replication forks as a distinct four-way DNA structure to protect against formation and exposure to toxic DNA lesions. So far, prevailing methods to measure RFs involve specialized electron microscopy precluding studies within their cellular context. Here we describe an in-situ method to quantitatively measure RFs by harnessing intrinsic bio-physical properties of this distinct DNA structure (RF-SIRF). RF-SIRF reveals that RFs accumulate at the nuclear periphery and form predominantly during early-mid S-phase of the cell cycle. Importantly, RFs are chromatinized and utilize an epigenetic replication stress code distinct from transcription that explains how DNA stress response proteins are recruited to RFs. Collectively, RF-SIRF enables robust, quantitative, temporal and spatial analyses of RFs and associated proteomics, empowering advanced cellular investigations of DNA replication stress responses.

Abstract Fanconi Anemia (FA) is a prototypic genetic disease signified by heterogeneous phenotypes including cancer, bone marrow failure, short stature, congenital abnormalities, infertility, sub-mendelian birth rate, genome instability and high cellular sensitivity to cancer therapeutics1-4. Clinical diagnosis is confirmed by identifying biallelic, homo- or hemizygous mutations in any one of twenty-three FANC genes1,5. Puzzlingly, inactivation of one single Fanc gene in mice fails to faithfully model the human disease manifestations6-8. We here delineate a preclinical Fanc mouse model with mutations in two genes, Fancd1/Brca2 and Fanco/Rad51c, that recapitulates the severity and heterogeneity of the human disease manifestations including death by cancer at young age. Surprisingly, these grave phenotypes cannot be explained by the sum of phenotypes seen in mice with single gene inactivation, which are unremarkable. In contrast to expectations from classic epistasis analysis of genetic pathways, the data instead reveal an unexpected functional synergism of polygenic Fanc mutations. Importantly in humans, whole exome sequencing uncovers that FANC co-mutation in addition to the identified inactivating FANC gene mutation is a frequent event in FA patients. Collectively, the data establish a concept of polygenic stress as an important contributor to disease manifestations, with implications for molecular diagnostics. Competing Interest Statement The authors have declared no competing interest.

Classically, p53 tumor-suppressor acts in transcription, apoptosis, and cell-cycle arrest. Yet, replication-mediated genomic instability is integral to oncogenesis, and p53 mutations promote tumor progression and drug-resistance. By delineating human and murine separation-of-function p53 alleles, we find that p53 null and gain-of-function (GOF) mutations exhibit defects in restart of stalled or damaged DNA replication forks driving genomic instability independent of transcription activation. By assaying protein-DNA fork interactions in single cells, we unveil a p53-MLL3-enabled recruitment of MRE11 DNA replication restart nuclease. Importantly, p53 defects or depletion unexpectedly allow mutagenic RAD52 and POL pathways to hijack stalled forks, which we find reflected in p53 defective breast-cancer patient COSMIC mutational signatures. These data uncover p53 as a keystone regulator of replication homeostasis within a DNA restart network. Mechanistically, this has important implications for development of resistance in cancer therapy. Combined, these results define an unexpected role for p53 suppression of replication genome instability.