The efficient repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity. In mammalian cells, the nonhomologous end-joining process that represents the predominant repair pathway relies on the DNA-dependent protein kinase (DNA-PK) and the XRCC4-DNA ligase IV complex. Nonetheless, several in vitro and in vivo results indicate that mammalian cells use more than a single end-joining mechanism. While searching for a DNA-PK-independent end-joining activity, we found that the pretreatment of DNA-PK-proficient and -deficient rodent cells with an inhibitor of the poly(ADP-ribose) polymerase-1 enzyme (PARP-1) led to increased cytotoxicity of the highly efficient DNA double-strand breaking compound calicheamicin ␥1. In addition, the repair kinetics of the DSBs induced by calicheamicin ␥1 was delayed both in PARP-1-proficient cells pretreated with the PARP-1 inhibitor and in PARP-1-deficient cells. In order to get new insights into the mechanism of an alternative route for DSBs repair, we have established a new synapsis and end-joining two-step assay in vitro, operating on DSBs with either nuclear protein extracts or recombinant proteins. We found an end-joining activity independent of the DNA-PK/XRCC4-ligase IV complex but that actually required a novel synapsis activity of PARP-1 and the ligation activity of the XRCC1-DNA ligase III complex, proteins otherwise involved in the base excision repair pathway. Taken together, these results strongly suggest that a PARP-1-dependent DSBs end-joining activity may exist in mammalian cells. We propose that this mechanism could act as an alternative route of DSBs repair that complements the DNA-PK/XRCC4/ligase IV-dependent nonhomologous end-joining.DNA double-strand breaks (DSBs) 1 represent normal intermediates during physiological processes like meiosis or V(D)J recombination but also toxic lesions produced by collapsed DNA replication forks and by DNA-damaging agents such as ionizing radiation (IR) or reactive oxygen species. Repair of DSBs is critical for the maintenance of genomic integrity because unproperly repaired breaks can lead to cancer via chromosomal aberrations (1, 2).In eukaryotic cells, DSBs are repaired through two major pathways: homologous recombination (HR) and nonhomologous end-joining (NHEJ) (for reviews see Refs. 3-6). It is largely admitted that DSBs are repaired by NHEJ at least in the G 1 phase of the cell cycle, whereas HR operates in late S/G 2 (7). The NHEJ process requires several factors that recognize and bind the double-strand break, catalyze the synapsis of the broken ends, and then process and reseal the break (8, 9). The NHEJ pathway relies on a set of proteins comprising at least (i) a DNA end binding activity, the DNA-dependent protein kinase (DNA-PK) that consists of the catalytic subunit DNA-PKcs and the Ku70/Ku80 heterodimer (10, 11), and (ii) a DNA break resealing activity, the XRCC4-DNA ligase IV complex (12, 13).In order to gain insight into the NHEJ mechanism, both in vitro and in vivo approaches have ...

Although high-protein diets (HPDs) are frequently consumed for body-weight control, little is known about the consequences for gut microbiota composition and metabolic activity and for large intestine mucosal homeostasis. Moreover, the effects of HPDs according to the source of protein need to be considered in this context. The objective of this study was to evaluate the effects of the quantity and source of dietary protein on microbiota composition, bacterial metabolite production, and consequences for the large intestinal mucosa in humans. A randomized, double-blind, parallel-design trial was conducted in 38 overweight individuals who received a 3-wk isocaloric supplementation with casein, soy protein, or maltodextrin as a control. Fecal and rectal biopsy-associated microbiota composition was analyzed by 16S ribosomal DNA sequencing. Fecal, urinary, and plasma metabolomes were assessed by H-nuclear magnetic resonance. Mucosal transcriptome in rectal biopsies was determined with the use of microarrays. HPDs did not alter the microbiota composition, but induced a shift in bacterial metabolism toward amino acid degradation with different metabolite profiles according to the protein source. Correlation analysis identified new potential bacterial taxa involved in amino acid degradation. Fecal water cytotoxicity was not modified by HPDs, but was associated with a specific microbiota and bacterial metabolite profile. Casein and soy protein HPDs did not induce inflammation, but differentially modified the expression of genes playing key roles in homeostatic processes in rectal mucosa, such as cell cycle or cell death. This human intervention study shows that the quantity and source of dietary proteins act as regulators of gut microbiota metabolite production and host gene expression in the rectal mucosa, raising new questions on the impact of HPDs on the large intestine mucosa homeostasis. This trial was registered at clinicaltrials.gov as NCT02351297.

Epidemiology shows that red and processed meat intake is associated with an increased risk of colorectal cancer. Heme iron, heterocyclic amines, and endogenous N-nitroso compounds (NOC) are proposed to explain this effect, but their relative contribution is unknown. Our study aimed at determining, at nutritional doses, which is the main factor involved and proposing a mechanism of cancer promotion by red meat. The relative part of heme iron (1% in diet), heterocyclic amines (PhIP þ MeIQx, 50 þ 25 mg/kg in diet), and NOC (induced by NaNO 2 þ NaNO 3 ; 0.17 þ 0.23 g/L of drinking water) was determined by a factorial design and preneoplastic endpoints in chemically induced rats and validated on tumors in Min mice. The molecular mechanisms (genotoxicity, cytotoxicity) were analyzed in vitro in normal and Apc-deficient cell lines and confirmed on colon mucosa. Heme iron increased the number of preneoplastic lesions, but dietary heterocyclic amines and NOC had no effect on carcinogenesis in rats. Dietary hemoglobin increased tumor load in Min mice (control diet: 67 AE 39 mm 2 ; 2.5% hemoglobin diet: 114 AE 47 mm 2 , P ¼ 0.004). In vitro, fecal water from rats given hemoglobin was rich in aldehydes and was cytotoxic to normal cells, but not to premalignant cells. The aldehydes 4-hydroxynonenal and 4-hydroxyhexenal were more toxic to normal versus mutated cells and were only genotoxic to normal cells. Genotoxicity was also observed in colon mucosa of mice given hemoglobin. These results highlight the role of heme iron in the promotion of colon cancer by red meat and suggest that heme iron could initiate carcinogenesis through lipid peroxidation. Cancer Res; 75(5); 870-9. Ó2015 AACR.