Cells possess an armamentarium of DNA repair pathways to counter DNA damage and prevent mutation. Here we use C. elegans whole genome sequencing to systematically quantify the contributions of these factors to mutational signatures. We analyse 2,717 genomes from wild-type and 53 DNA repair defective backgrounds, exposed to 11 genotoxins, including UV-B and ionizing radiation, alkylating compounds, aristolochic acid, aflatoxin B1, and cisplatin. Combined genotoxic exposure and DNA repair deficiency alters mutation rates or signatures in 41% of experiments, revealing how different DNA alterations induced by the same genotoxin are mended by separate repair pathways. Error-prone translesion synthesis causes the majority of genotoxin-induced base substitutions, but averts larger deletions. Nucleotide excision repair prevents up to 99% of point mutations, almost uniformly across the mutation spectrum. Our data show that mutational signatures are joint products of DNA damage and repair and suggest that multiple factors underlie signatures observed in cancer genomes.

Macrophages reside in the body cavities where they maintain serosal homeostasis and provide immune surveillance. Peritoneal macrophages are implicated in the etiology of pathologies including peritonitis, endometriosis, and metastatic cancer; thus, understanding the factors that govern their behavior is vital. Using a combination of fate mapping techniques, we have investigated the impact of sex and age on murine peritoneal macrophage differentiation, turnover, and function. We demonstrate that the sexually dimorphic replenishment of peritoneal macrophages from the bone marrow, which is high in males and very low in females, is driven by changes in the local microenvironment that arise upon sexual maturation. Population and single-cell RNA sequencing revealed marked dimorphisms in gene expression between male and female peritoneal macrophages that was, in part, explained by differences in composition of these populations. By estimating the time of residency of different subsets within the cavity and assessing development of dimorphisms with age and in monocytopenic Ccr2^−/− mice, we demonstrate that key sex-dependent features of peritoneal macrophages are a function of the differential rate of replenishment from the bone marrow, whereas others are reliant on local microenvironment signals. We demonstrate that the dimorphic turnover of peritoneal macrophages contributes to differences in the ability to protect against pneumococcal peritonitis between the sexes. These data highlight the importance of considering both sex and age in susceptibility to inflammatory and infectious diseases.

Summary: The mechanism of bacteriophage DNA injection is poorly understood, often considered a simple process, driven merely by the packing pressure inside the capsid. In contrast to the well‐established DNA packaging mechanism of Bacillus subtilis phage Ø29, that involves a molecular motor formed by the connector and a viral ATPase, nothing is known about its DNA injection into the cell. We have studied this process measuring DNA binding of p6, a viral genome organization protein. The linear DNA penetrates with a right‐left polarity, in a two‐step process. In the first step ∼65% of the genome is pushed into the cell most probably by the pressure built inside the viral capsid. Thus, synthesis of viral proteins from the right early operon is allowed. This step is controlled, probably by bacterial protein(s) that slow down DNA entry. In the second step at least one of the viral early proteins, p17, participates in the molecular machinery that pulls the remaining DNA inside the cell. Both steps are energy‐dependent, as treatment of cells with azide overrides the whole mechanism, leading to a deregulated, passive entry of DNA.

DNA replication is sensitive to damage in the template. To bypass lesions and complete replication, cells activate recombination‐mediated (error‐free) and translesion synthesis‐mediated (error‐prone) DNA damage tolerance pathways. Crucial for error‐free DNA damage tolerance is template switching, which depends on the formation and resolution of damage‐bypass intermediates consisting of sister chromatid junctions. Here we show that a chromatin architectural pathway involving the high mobility group box protein Hmo1 channels replication‐associated lesions into the error‐free DNA damage tolerance pathway mediated by Rad5 and PCNA polyubiquitylation, while preventing mutagenic bypass and toxic recombination. In the process of template switching, Hmo1 also promotes sister chromatid junction formation predominantly during replication. Its C‐terminal tail, implicated in chromatin bending, facilitates the formation of catenations/hemicatenations and mediates the roles of Hmo1 in DNA damage tolerance pathway choice and sister chromatid junction formation. Together, the results suggest that replication‐associated topological changes involving the molecular DNA bender, Hmo1, set the stage for dedicated repair reactions that limit errors during replication and impact on genome stability.

The association of increased levels of tumour-infiltrating gamma-delta (γδ) T cells with favorable prognosis across many cancer types and their ability to recognize stress antigens in an MHC unrestricted manner has led to an increased interest in exploiting them for cancer immunotherapy. We performed single-cell RNA sequencing (scRNA-seq) of peripheral blood γδ T cells from healthy adult donors and from fresh tumour biopsies of breast cancer patients. We identified five γδ T cells subtypes in blood and three subtypes of γδ T cells in breast tumour. These subtypes differed in the expression of genes contributing to effector functions such as antigen presentation, cytotoxicity, and IL17A and IFNγ production. Compared with the blood γδ T cells, the breast tumour-infiltrating γδ T cells were more activated, expressed higher levels of cytotoxic genes, yet were immunosuppressed. One subtype in the breast tumour that was IFNγ-positive had no obvious similarity to any of the subtypes observed in the blood γδ T cell and was the only subtype associated with improved overall survival of breast cancer patients. Taken together, our study has identified markers of subtypes of human blood γδ T cells and uncovered a tumour-infiltrating γδ T cells subtype associated improved overall cancer survival.