BRCA1 protects against its own fragility.

Deshpande et al. (2022) demonstrate that BRCA1, a tumor suppressor tasked with protecting the genome, is encoded by a gene that is intrinsically fragile.

Disruption of DNA polymerase ζ engages an innate immune response.

In mammalian cells, specialized DNA polymerase ζ (pol ζ) contributes to genomic stability during normal DNA replication. Disruption of the catalytic subunit Rev3l is toxic and results in constitutive chromosome damage, including micronuclei. As manifestations of this genomic stress are unknown, we examined the transcriptome of pol ζ-defective cells by RNA sequencing (RNA-seq). Expression of 1,117 transcripts is altered by ≥4-fold in Rev3l-disrupted cells, with a pattern consistent with an induction of an innate immune response. Increased expression of interferon-stimulated genes at the mRNA and protein levels in pol ζ-defective cells is driven by the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-signaling partner stimulator of interferon genes (STING) pathway. Expression of key interferon-stimulated chemokines is elevated in basal epithelial mouse skin cells with a disruption of Rev3l. These results indicate that the disruption of pol ζ may simultaneously increase sensitivity to genotoxins and potentially engage parts of the innate immune response, which could add an additional benefit to targeting pol ζ in cancer therapies.

DNA polymerase ζ in DNA replication and repair.

Here, we survey the diverse functions of DNA polymerase ζ (pol ζ) in eukaryotes. In mammalian cells, REV3L (3130 residues) is the largest catalytic subunit of the DNA polymerases. The orthologous subunit in yeast is Rev3p. Pol ζ also includes REV7 subunits (encoded by Rev7 in yeast and MAD2L2 in mammalian cells) and two subunits shared with the replicative DNA polymerase, pol δ. Pol ζ is used in response to circumstances that stall DNA replication forks in both yeast and mammalian cells. The best-examined situation is translesion synthesis at sites of covalent DNA lesions such as UV radiation-induced photoproducts. We also highlight recent evidence that uncovers various roles of pol ζ that extend beyond translesion synthesis. For instance, pol ζ is also employed when the replisome operates sub-optimally or at difficult-to-replicate DNA sequences. Pol ζ also participates in repair by microhomology mediated break-induced replication. A rev3 deletion is tolerated in yeast but Rev3l disruption results in embryonic lethality in mice. Inactivation of mammalian Rev3l results in genomic instability and invokes cell death and senescence programs. Targeting of pol ζ function may be a useful strategy in cancer therapy, although chromosomal instability associated with pol ζ deficiency must be considered.

Constitutive role of the Fanconi anemia D2 gene in the replication stress response.

In response to DNA cross-linking damage, the Fanconi anemia (FA) core complex activates the FA pathway by monoubiquitinating Fanconi anemia complementation group D2 (FANCD2) for the initiation of the nucleolytic processing of the DNA cross-links and stabilization of stalled replication forks. Given that all the classic FA proteins coordinately monoubiquitinate FANCD2, it is unclear why losses of individual classic FA genes yield varying cellular sensitivities to cross-linking damage. To address this question, we generated cellular knock-out models of FA core complex components and FANCD2 and found that FANCD2-null mutants display higher levels of spontaneous chromosomal damage and hypersensitivity to replication-blocking lesions than Fanconi anemia complementation group L (FANCL)-null mutants, suggesting that FANCD2 provides a basal level of DNA protection countering endogenous lesions in the absence of monoubiquitination. FANCD2's ubiquitination-independent function is likely involved in optimized recruitment of nucleolytic activities for the processing and protection of stressed replication forks. Our results reveal that FANCD2 has a ubiquitination-independent role in countering endogenous levels of replication stress, a function that is critical for the maintenance of genomic stability.

Expression and Structural Analyses of Human DNA Polymerase θ (POLQ).

DNA polymerase theta (pol θ) is an evolutionarily conserved protein encoded by the POLQ gene in mammalian genomes. Pol θ is the defining enzyme for a pathway of DSB repair termed "alternative end-joining" (altEJ) or "theta-mediated end-joining." This pathway contributes significantly to the radiation resistance of mammalian cells. It also modulates accuracy in repair of breaks that occur at stalled DNA replication forks, during diversification steps of the mammalian immune system, during repair of CRISPR-Cas9, and in many DNA integration events. Pol θ is a potentially important clinical target, particularly for cancers deficient in other break repair strategies. The enzyme is uniquely able to mediate joining of single-stranded 3' ends. Because of these unusual biochemical properties and its therapeutic importance, it is essential to study structures of pol θ bound to DNA. However, challenges for expression and purification are presented by the large size of pol θ (2590 residues in humans) and unusual juxtaposition of domains (a helicase-like domain and distinct DNA polymerase, separated by a region predicted to be largely disordered). Here we summarize work on the expression and purification of the full-length protein, and then focus on the design, expression, and purification of an active C-terminal polymerase fragment. The generation of this active construct was nontrivial and time consuming. Almost all published biochemical work to date has been performed with this domain fragment. Strategies to obtain and improve crystals of a ternary pol θ complex (enzyme:DNA:nucleotide) are also presented, along with key elements of the structure.

Does Eulemur cinereiceps exist? Preliminary evidence from genetics and ground surveys in southeastern Madagascar.

Although appearing in the literature as early as 1890, the brown lemur form Eulemur cinereiceps has recently resurfaced as a potentially valid taxon, distinct from neighboring, presumably closely related species such as white-collared lemurs (Eulemur albocollaris). We propose two scenarios for the potential separation of E. cinereiceps and E. albocollaris: (1) coastal and interior populations represent two distinct taxa and (2) the coastal population north of the Manampatrana River (the locality for purported museum specimens of E. cinereiceps) represents a distinct species from E. albocollaris found south of the river and in the interior escarpment forests. We tested these hypotheses using data from ground surveys and genetic sampling. Surveys were conducted in coastal forest fragments both north and south of the Manampatrana River in July-August 2006. Genetic samples were collected at two coastal sites and one interior forest. We used maximum parsimony, maximum likelihood, and neighbor-joining analyses on mitochondrial DNA regions to determine if populations from different sites clustered into diagnosable clades. Results from field surveys confirmed the presence of forms commonly referred to as E. albocollaris at the two southern coastal forests; no consistent phenotypic differences across sites were observed. All genetic analyses yielded identical results: coastal and interior populations do not cluster into separate groups, thus rejecting the first hypothesis. Eulemur species and all other day-active lemurs have apparently been extirpated from coastal forests north of the Manampatrana. Owing to the absence of lemurs from the northern coastal localities, we could not conclusively support or reject the second scenario. However, based on examination of the original plates and museum specimens, as well as the biogeographic patterns typical of this region, we strongly suspect that all populations from this area belong to a single species. We conclude with remarks regarding the apparent priority of E. cinereiceps for this taxon.