A novel function for the Mre11-Rad50-Xrs2 complex in base excision repair

The Mre11/Rad50/Xrs2 (MRX) complex in Saccharomyces cerevisiae has well-characterized functions in DNA double-strand break processing, checkpoint activation, telomere length maintenance and meiosis. In this study, we demonstrate an involvement of the complex in the base excision repair (BER) pathway. We studied the repair of methyl-methanesulfonate-induced heat-labile sites in chromosomal DNA in vivo and the in vitro BER capacity for the repair of uracil- and 8-oxoG-containing oligonucleotides in MRX-deficient cells. Both approaches show a clear BER deficiency for the xrs2 mutant as compared to wildtype cells. The in vitro analyses revealed that both subpathways, long-patch and short-patch BER, are affected and that all components of the MRX complex are similarly important for the new function in BER. The investigation of the epistatic relationship of XRS2 to other BER genes suggests a role of the MRX complex downstream of the AP-lyases Ntg1 and Ntg2. Analysis of individual steps in BER showed that base recognition and strand incision are not affected by the MRX complex. Reduced gap-filling activity and the missing effect of aphidicoline treatment, an inhibitor for polymerases, on the BER efficiency indicate an involvement of the MRX complex in providing efficient polymerase activity

A Key Role for E-cadherin in Intestinal Homeostasis and Paneth Cell Maturation

E-cadherin is a major component of adherens junctions. Impaired expression of E-cadherin in the small intestine and colon has been linked to a disturbed intestinal homeostasis and barrier function. Down-regulation of E-cadherin is associated with the pathogenesis of infections with enteropathogenic bacteria and Crohn's disease. To genetically clarify the function of E-cadherin in intestinal homeostasis and maintenance of the epithelial defense line, the Cdh1 gene was conditionally inactivated in the mouse intestinal epithelium. Inactivation of the Cdh1 gene in the small intestine and colon resulted in bloody diarrhea associated with enhanced apoptosis and cell shedding, causing life-threatening disease within 6 days. Loss of E-cadherin led cells migrate faster along the crypt-villus axis and perturbed cellular differentiation. Maturation and positioning of goblet cells and Paneth cells, the main cell lineage of the intestinal innate immune system, was severely disturbed. The expression of anti-bacterial cryptidins was reduced and mice showed a deficiency in clearing enteropathogenic bacteria from the intestinal lumen. These results highlight the central function of E-cadherin in the maintenance of two components of the intestinal epithelial defense: E-cadherin is required for the proper function of the intestinal epithelial lining by providing mechanical integrity and is a prerequisite for the proper maturation of Paneth and goblet cells

Loss of differentiated cells following loss of E-cadherin.

<p>(A and B) Staining of absorptive enterocytes for villin. Reduced staining intensity and disruption of the epithelial lining (arrowheads) in the small intestine (A) and colon (B) of Cre⁺<i>Cdh1</i>^fl/fl (KO) mice as compared to controls (WT). (C and D) PAS staining revealed a strong reduction of goblet cells (arrowheads) in the colon (C) and small intestine (D) of Cre⁺<i>Cdh1</i>^fl/fl mice four days after start of induction of recombination as compared to control mice. (E and F) Paneth cells were identified by immunostaining for lysozyme (E) and MMP7 (F). After five days of tamoxifen treatment, Paneth cells remained confined to the base of the crypt in control mice, but were distributed throughout the crypt-villus axis in Cre⁺<i>Cdh1</i>^fl/fl mice (arrowheads). Bars in A–F, 100 µm.</p

E-cadherin deficiency abrogates localization and differentiation of cells.

<p>(A and B) PAS staining of small intestine (A) and colon (B) in control mice (WT) and Cre⁺<i>Cdh1</i>^fl/fl mice (KO) analyzed on day 12 following treatment with tamoxifen on days 1, 2, 5, and 8 revealed a reduction of the number of goblet cells and the appearance of large atypical goblet cells (arrowheads). (C and D) Staining for lysozyme (C) and MMP7 (D) in control mice (WT) and Cre⁺<i>Cdh1</i>^fl/fl mice (KO) analyzed on day 12 following treatment with tamoxifen as described above revealed a reduction of Paneth cells at the base of the crypts and the appearance of large positively stained cells in the villi (arrowheads). (E and F) BrdU staining to evaluate cell migration in control (E) and Cre⁺<i>Cdh1</i>^fl/fl (F) mice 1h (left) and 24h (right) after injection of BrdU. After 24h, BrdU-positive cells were still confined to the lower parts of the villi in control mice, but positive cells were found throughout the entire crypt-villus axis in Cre⁺<i>Cdh1</i>^fl/fl mice. (G and H) Position of BrdU-positive cells at 1 h (black bars) and 24 h (open bars) within the crypt-villus axis of control and Cre⁺<i>Cdh1</i>^fl/fl mice. Position 0 represents the base of the crypt. Data were obtained by evaluating 20 crypt-villus units per mouse (n = 2 animals/group). Bars in A and B, 100 µm.</p

Induction of cell death and loss of adherens junctions and desmosomes upon loss of E-cadherin.

<p>(A and B) Immunostaining of small intestine (A) and colon (B) for cleaved caspase 3 in control (WT) and Cre⁺<i>Cdh1</i>^fl/fl (KO) mice treated with tamoxifen for 5 days was employed to identify apoptotic cells (arrowheads). (C) Quantitative analysis of apoptotic cells. 20 crypt-villus units in small intestine and 20 crypts in colon per animal were analyzed at the indicated time points (n = 4 mice/group). The rate of apoptotic cells is expressed as a percentage of total cell numbers and mean values and standard error means are shown (*, <i>P</i><0.005). (D) Transmission electron microscopy of the apical junctional complex of control (WT) and Cre⁺<i>Cdh1</i>^fl/fl (KO) mice. The intercellular space, microvilli (mv) and cellular substructures appeared unaltered. Compared to control mice, the junctional complex of Cre⁺<i>Cdh1</i>^fl/fl mice had preserved tight junctions (white arrowhead), but lacked adherens junctions with <i>rete terminale</i> (black arrowhead) and desmosomes (black arrow). Bars in A and C, 100 µm, in D 0.2 µm.</p

Impairment of bacterial defense in mice deficient for E-cadherin.

<p>Increase of bioluminescence in the Peyer's patches in Cre⁺<i>Cdh</i>1^fl/fl mice (A, right panel) compared to control mice (A, left panel) 5 days after oral infection with with 10⁹ CFU of bioluminescent <i>Yersinia enterocolitica</i>. Colonization of small intestine (B), and Peyer's Patches (C) of Cre⁺<i>Cdh1</i>^fl/fl and control mice (Cre⁺<i>Cdh1</i>^wt/fl). CFU for each mouse is shown and bars represent the median value. The limit of detection for yersiniae was 10 CFU for Peyer's Patches and 50 CFU for small intestine. Statistical significance is indicated by asteriks.</p

Impaired expression of E-cadherin results in elongation of crypts and villi.

<p>H&E staining of Cre⁺<i>Cdh1</i>^fl/fl (KO) and control mice (WT) on day 12 after induction of recombination on days 1, 2, 5, and 8, revealed milder changes in the epithelial architecture of small intestine (A) and colon (B) with elongation of crypts and disorganization of the cellular order. Immunostaining for E-cadherin revealed a reduction in E-cadherin expression 12 days after start of recombination in KO compared to WT mice in small intestine (C) and colon (D). Bars, 100 µm.</p

Loss of E-cadherin results in disturbed maturation of Paneth cells.

<p>(A–B) Staining of ultra thin sections (A) and transmission electron microscopy (B) revealed a reduced number of Paneth cells at the base of the crypts (arrowheads), altered Paneth cell granule morphology, and reduced numbers and smaller diameters of Paneth cell granules in Cre⁺<i>Cdh1</i>^fl/fl (left panels) compared to control mice (right panels). Bars in A and B, 20 µm and 2.5 µm, respectively. (C) Quantitative analysis of ultra thin sections revealed a reduced number of Paneth cells per crypt and a significant increase in the fraction of cryts with <3 Paneth cells in recombined mice (100 crypts analyzed per mouse for n = 4 mice per group. *, <i>P</i><0.005. Measurement of Paneth cell granules in the same mice revealed an increase in small granules in E-cadherin deficient mice. <i>P</i><0.005. (D) Expression of cryptidin 1 and 4, lysozyme and Muc2 mRNA was determined by quantitative RT-PCR in the intestinal epithelia of control (n = 5) and Cre⁺<i>Cdh1</i>^fl/fl (n = 5) mice. (E–L) Co-immunofluorescence for Muc2 (E, I), lysozyme (F, J), E-cadherin (G, K) in control (E–H) and Cre⁺<i>Cdh1</i>^fl/fl mice (I–L). (H, L) Fusion of pictures. Bars in E–L, 10 µm.</p