We targeted the locus encoding the cyclin-dependent kinase 2 (CDK2) by homologous recombination in mouse embryonic stem (ES) cells. Embryonic fibroblasts lacking CDK2 proliferate normally and become immortal after continuous passage in culture. Elimination of a conditional Cdk2 allele in immortal cells does not have a significant effect on proliferation. Cdk2-/- mice are viable and survive for up to two years, indicating that CDK2 is also dispensable for proliferation and survival of most cell types. But CDK2 is essential for completion of prophase I during meiotic cell division in male and female germ cells, an unforeseen role for this cell cycle kinase.

53BP1 is a chromatin-binding protein that regulates the repair of DNA double-strand breaks by suppressing the nucleolytic resection of DNA termini. This function of 53BP1 requires interactions with PTIP and RIF1, the latter of which recruits REV7 (also known as MAD2L2) to break sites. How 53BP1-pathway proteins shield DNA ends is currently unknown, but there are two models that provide the best potential explanation of their action. In one model the 53BP1 complex strengthens the nucleosomal barrier to end-resection nucleases, and in the other 53BP1 recruits effector proteins with end-protection activity. Here we identify a 53BP1 effector complex, shieldin, that includes C20orf196 (also known as SHLD1), FAM35A (SHLD2), CTC-534A2.2 (SHLD3) and REV7. Shieldin localizes to double-strand-break sites in a 53BP1- and RIF1-dependent manner, and its SHLD2 subunit binds to single-stranded DNA via OB-fold domains that are analogous to those of RPA1 and POT1. Loss of shieldin impairs non-homologous end-joining, leads to defective immunoglobulin class switching and causes hyper-resection. Mutations in genes that encode shieldin subunits also cause resistance to poly(ADP-ribose) polymerase inhibition in BRCA1-deficient cells and tumours, owing to restoration of homologous recombination. Finally, we show that binding of single-stranded DNA by SHLD2 is critical for shieldin function, consistent with a model in which shieldin protects DNA ends to mediate 53BP1-dependent DNA repair.

We have unveiled a synthetic lethal interaction between K-Ras oncogenes and Cdk4 in a mouse tumor model that closely recapitulates human non-small cell lung carcinoma (NSCLC). Ablation of Cdk4, but not Cdk2 or Cdk6, induces an immediate senescence response only in lung cells that express an endogenous K-Ras oncogene. No such response occurs in lungs expressing a single Cdk4 allele or in other K-Ras-expressing tissues. More importantly, targeting Cdk4 alleles in advanced tumors detectable by computed tomography scanning also induces senescence and prevents tumor progression. These observations suggest that robust and selective pharmacological inhibition of Cdk4 may provide therapeutic benefit for NSCLC patients carrying K-RAS oncogenes.

The response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity and gametogenesis. In order to provide an unbiased and global view of the DNA damage response in human cells, we undertook 28 CRISPR/Cas9 screens against 25 genotoxic agents in the retinal pigment epithelium-1 (RPE1) cell line. These screens identified 840 genes whose loss causes either sensitivity or resistance to DNA damaging agents. Mining this dataset, we uncovered that ERCC6L2, which is mutated in a bone-marrow failure syndrome, codes for a canonical non-homologous end-joining pathway factor; that the RNA polymerase II component ELOF1 modulates the response to transcription-blocking agents and that the cytotoxicity of the G-quadruplex ligand pyridostatin involves trapping topoisomerase II on DNA. This map of the DNA damage response provides a rich resource to study this fundamental cellular system and has implications for the development and use of genotoxic agents in cancer therapy.

In recent years, our understanding of the mechanisms underlying colorectal carcinogenesis has vastly expanded. Underlying inflammation within the intestine, diet, and most recently, the gut microbiota, have been demonstrated to influence the development of colorectal cancer. However, since cancer is ultimately a genetic disease, these factors are thought to create genotoxic stress within the intestinal environment to promote genetic and epigenetic alterations leading to cancer. In this review, we will focus on how gut microbes intersect with inflammation, diet, and host genetics to influence the development of colon cancer.

The generation of protective antibodies requires somatic hypermutation (SHM) and class-switch recombination (CSR) of immunoglobulin genes. Here we show that mice mutant for exonuclease 1 (Exo1), which participates in DNA mismatch repair (MMR), have decreased CSR and changes in the characteristics of SHM similar to those previously observed in mice mutant for the MMR protein Msh2. Exo1 is thus the first exonuclease shown to be involved in SHM and CSR. The phenotype of Exo1(-/-) mice and the finding that Exo1 and Mlh1 are physically associated with mutating variable regions support the idea that Exo1 and MMR participate directly in SHM and CSR.

The production of high-affinity protective antibodies requires somatic hypermutation (SHM) of the antibody variable (V)-region genes. SHM is characterized by a high frequency of point mutations that occur only during the centroblast stage of B-cell differentiation. Activation-induced cytidine deaminase (AID), which is expressed specifically in germinal-centre centroblasts, is required for this process, but its exact role is unknown. Here we show that AID is required for SHM in the centroblast-like Ramos cells, and that expression of AID is sufficient to induce SHM in hybridoma cells, which represent a later stage of B-cell differentiation that does not normally undergo SHM. In one hybridoma, mutations were exclusively in G*C base pairs that were mostly within RGYW or WRCY motifs, suggesting that AID has primary responsibility for mutations at these nucleotides. The activation of SHM in hybridomas indicates that AID does not require other centroblast-specific cofactors to induce SHM, suggesting either that it functions alone or that the factors it requires are expressed at other stages of B-cell differentiation.

The largest mucosal surface in the body is in the gastrointestinal (GI) tract, a location that is heavily colonized by normally harmless microbes. A key mechanism required for maintaining a homeostatic balance between this microbial burden and the lymphocytes that densely populate the GI tract is the production and trans-epithelial transport of poly-reactive IgA1. Within the mucosal tissues, B cells respond to cytokines, sometimes in the absence of T cell help, undergo class switch recombination (CSR) of their Immunoglobulin (Ig) receptor to IgA, and differentiate to become plasma cells (PC)2. However, IgA-secreting PC likely have additional attributes that are needed for coping with the tremendous bacterial load in the GI tract. Here we report that IgA+ PC also produce the anti-microbial mediators TNFα and iNOS, and express many molecules that are commonly associated with monocyte/granulocytic cell types. The development of iNOS-producing IgA+ PC can be recapitulated in vitro in the presence of gut stroma, and the acquisition of this multi-functional phenotype in vivo and in vitro relies on microbial co-stimulation. Deletion of TNFα and iNOS in B-lineage cells resulted in a reduction in IgA production, altered diversification of the gut microbiota and poor clearance of a gut-tropic pathogen. These findings reveal a novel adaptation to maintaining homeostasis in the gut, and extend the repertoire of protective responses exhibited by some B lineage cells.