While biallelic mutations in the PALB2 tumor suppressor cause Fanconi anemia subtype FA-N, monoallelic mutations predispose to breast and familial pancreatic cancer. Although hundreds of missense variants in PALB2 have been identified in patients to date, only a few have clear functional and clinical relevance. Herein, we investigate the effects of 44 PALB2 variants of uncertain significance found in breast cancer patients and provide detailed analysis by systematic functional assays. Our comprehensive functional analysis reveals two hotspots for potentially deleterious variations within PALB2, one at each terminus. PALB2 N-terminus variants p.P8L [c.23C>T], p.Y28C [c.83A>G], and p.R37H [c.110G>A] compromised PALB2-mediated homologous recombination. At the C-terminus, PALB2 variants p.L947F [c.2841G>T], p.L947S [c.2840T>C], and most strikingly p.T1030I [c.3089C>T] and p.W1140G [c.3418T>C], stood out with pronounced PARP inhibitor sensitivity and cytoplasmic accumulation in addition to marked defects in recruitment to DNA damage sites, interaction with BRCA2 and homologous recombination. Altogether, our findings show that a combination of functional assays is necessary to assess the impact of germline missense variants on PALB2 function, in order to guide proper classification of their deleteriousness.

To identify candidate variants in RAD51C and RAD51D ovarian cancer (OC) predisposing genes by investigating French Canadians (FC) exhibiting unique genetic architecture. Candidates were identified by whole exome sequencing analysis of 17 OC families and 53 early-onset OC cases. Carrier frequencies were determined by the genetic analysis of 100 OC or HBOC families, 438 sporadic OC cases and 1025 controls. Variants of unknown function were assayed for their biological impact and/or cellular sensitivity to olaparib. RAD51C c.414G>C;p.Leu138Phe and c.705G>T;p.Lys235Asn and RAD51D c.137C>G;p.Ser46Cys, c.620C>T;p.Ser207Leu and c.694C>T;p.Arg232Ter were identified in 17.6% of families and 11.3% of early-onset cases. The highest carrier frequency was observed in OC families (1/44, 2.3%) and sporadic cases (15/438, 3.4%) harbouring RAD51D c.620C>T versus controls (1/1025, 0.1%). Carriers of c.620C>T (n = 7), c.705G>T (n = 2) and c.137C>G (n = 1) were identified in another 538 FC OC cases. RAD51C c.705G>T affected splicing by skipping exon four, while RAD51D p.Ser46Cys affected protein stability and conferred olaparib sensitivity. Genetic and functional assays implicate RAD51C c.705G>T and RAD51D c.137C>G as likely pathogenic variants in OC. The high carrier frequency of RAD51D c.620C>T in FC OC cases validates previous findings. Our findings further support the role of RAD51C and RAD51D in hereditary OC.

Selenium is an essential trace element important for human health. A balanced intake is, however, crucial to maximize the health benefits of selenium. At physiological concentrations, selenium mediates antioxidant, anti-inflammatory, and prosurvival actions. However, supra-nutritional selenium intake was associated with increased diabetes risk leading potentially to endothelial dysfunction, the initiating step in atherosclerosis. High selenium causes apoptosis in cancer cells via endoplasmic reticulum (ER) stress, a mechanism also implicated in endothelial dysfunction. Nonetheless, whether ER stress drives selenium-induced endothelial dysfunction, remains unknown. Here, we investigated the effects of increasing concentrations of selenium on endothelial cells. High selenite reduced nitric oxide bioavailability and impaired angiogenesis. High selenite also induced ER stress, increased reactive oxygen species (ROS) production, and apoptosis. Pretreatment with the chemical chaperone, 4-phenylbutyrate, prevented the toxic effects of selenium. Our findings support a model where high selenite leads to endothelial dysfunction through activation of ER stress and increased ROS production. These results highlight the importance of tailoring selenium supplementation to achieve maximal health benefits and suggest that prophylactic use of selenium supplements as antioxidants may entail risk.

DNA alkylation damage is repaired by base excision repair (BER) initiated by alkyladenine DNA glycosylase (AAG). Despite its role in DNA repair, AAG-initiated BER promotes cytotoxicity in a process dependent on poly (ADP-ribose) polymerase-1 (PARP-1); a NAD + -consuming enzyme activated by strand break intermediates of the AAG-initiated repair process. Importantly, PARP-1 activation has been previously linked to impaired glycolysis and mitochondrial dysfunction. However, whether alkylation affects cellular metabolism in the absence of AAG-mediated BER initiation is unclear. To address this question, we temporally profiled repair and metabolism in wild-type and Aag −/− cells treated with the alkylating agent methyl methanesulfonate (MMS). We show that, although Aag −/− cells display similar levels of alkylation-induced DNA breaks as wild type, PARP-1 activation is undetectable in AAG-deficient cells. Accordingly, Aag −/− cells are protected from MMS-induced nAD + depletion and glycolysis inhibition. MMS-induced mitochondrial dysfunction, however, is AAGindependent. Furthermore, treatment with FK866, a selective inhibitor of the NAD + salvage pathway enzyme nicotinamide phosphoribosyltransferase (NAMPT), synergizes with MMS to induce cytotoxicity and Aag −/− cells are resistant to this combination FK866 and MMS treatment. Thus, AAG plays an important role in the metabolic response to alkylation that could be exploited in the treatment of conditions associated with NAD + dysregulation.DNA base damage is unavoidable and abundant, arising from hydrolysis, oxidation, deamination and alkylation reactions 1 . Base damage arises both as a by-product of cellular metabolism and by exposure to environmental agents and has been associated with several pathologies such as cancer, chronic inflammation and neurodegeneration 2,3 . Chiefly dealing with endogenous DNA base lesions is the base excision repair (BER) pathway. Initiation of the BER pathway occurs via the removal of a damaged base by one of many glycosylases displaying substrate specificities. Base excision leads to the formation of abasic sites and strand breaks that are subsequently processed by downstream enzymes so that DNA synthesis and ligation can take place 4 (Fig. 1A). Since many of the

In a cross-sectional study of women in a nursing team at a university hospital in southern Brazil, we studied DNA damage, salivary cortisol levels, and cognition. DNA damage was measured in blood leukocytes with the comet assay and the micronucleus test. Salivary cortisol levels were determined upon waking, 30 min later, and at bedtime. Cognition was evaluated according to the Stroop, Digit span and Word span tests. Cortisol levels on waking up were associated negatively with the number of years the employee worked at the institution and positively with the DNA damage in comet assay. Cognitive scores were lower when the cortisol levels were low at awakening and high at bedtime; and were associated positively with educational level. Cortisol status may influence overall health as well as essential work skills, such as attention.

Alkylating agents damage DNA and proteins and are widely used in cancer chemotherapy. While cellular responses to alkylation-induced DNA damage have been explored, knowledge of how alkylation affects global cellular stress responses is sparse. Here, we examined the effects of the alkylating agent methylmethane sulfonate (MMS) on gene expression in mouse liver, using mice deficient in alkyladenine DNA glycosylase (Aag), the enzyme that initiates the repair of alkylated DNA bases. MMS induced a robust transcriptional response in wild-type liver that included markers of the endoplasmic reticulum (ER) stress/unfolded protein response (UPR) known to be controlled by XBP1, a key UPR effector. Importantly, this response is significantly reduced in the Aag knockout. To investigate how AAG affects alkylation-induced UPR, the expression of UPR markers after MMS treatment was interrogated in human glioblastoma cells expressing different AAG levels. Alkylation induced the UPR in cells expressing AAG; conversely, AAG knockdown compromised UPR induction and led to a defect in XBP1 activation. To verify the requirements for the DNA repair activity of AAG in this response, AAG knockdown cells were complemented with wild-type Aag or with an Aag variant producing a glycosylase-deficient AAG protein. As expected, the glycosylase-defective Aag does not fully protect AAG knockdown cells against MMS-induced cytotoxicity. Remarkably, however, alkylation-induced XBP1 activation is fully complemented by the catalytically inactive AAG enzyme. This work establishes that, besides its enzymatic activity, AAG has noncanonical functions in alkylation-induced UPR that contribute to cellular responses to alkylation.

The tumour suppressor PALB2 stimulates RAD51-mediated homologous recombination (HR) repair of DNA damage, whilst its steady-state association with active genes protects these loci from replication stress. Here, we report that the lysine acetyltransferases 2A and 2B (KAT2A/2B, also called GCN5/PCAF), two well-known transcriptional regulators, acetylate a cluster of seven lysine residues (7K-patch) within the PALB2 chromatin association motif (ChAM) and, in this way, regulate context-dependent PALB2 binding to chromatin. In unperturbed cells, the 7K-patch is targeted for KAT2A/2B-mediated acetylation, which in turn enhances the direct association of PALB2 with nucleosomes. Importantly, DNA damage triggers a rapid deacetylation of ChAM and increases the overall mobility of PALB2. Distinct missense mutations of the 7K-patch render the mode of PALB2 chromatin binding, making it either unstably chromatin-bound (7Q) or randomly bound with a reduced capacity for mobilisation (7R). Significantly, both of these mutations confer a deficiency in RAD51 foci formation and increase DNA damage in S phase, leading to the reduction of overall cell survival. Thus, our study reveals that acetylation of the ChAM 7K-patch acts as a molecular switch to enable dynamic PALB2 shuttling for HR repair while protecting active genes during DNA replication.

Fanconi Anemia (FA) is a rare, genome instability-associated disease characterized by a deficiency in repairing DNA crosslinks, which are known to perturb several cellular processes, including DNA transcription, replication, and repair. Formaldehyde, a by-product of metabolism, is thought to drive FA by generating DNA interstrand crosslinks (ICLs) and DNA-protein crosslinks (DPCs). However, the impact of formaldehyde on global cellular pathways has not been investigated thoroughly. Herein, using a pangenomic CRISPR-Cas9 screen, we identify EXO1 as a critical regulator of formaldehyde-induced DNA lesions. We show that EXO1 knockout cell lines exhibit formaldehyde sensitivity leading to the accumulation of replicative stress, DNA double-strand breaks, and quadriradial chromosomes, a typical feature of FA. After formaldehyde exposure, EXO1 is recruited to chromatin, protects DNA replication forks from degradation, and functions in parallel with the FA pathway to promote cell survival. In vitro, EXO1-mediated exonuclease activity is proficient in removing DPCs. Collectively, we show that EXO1 limits replication stress and DNA damage to counteract formaldehyde-induced genome instability.