Context-Dependent Remodeling of Rad51–DNA Complexes by Srs2 Is Mediated by a Specific Protein–Protein Interaction

The yeast Srs2 helicase removes Rad51 nucleoprotein filaments from single-stranded DNA (ssDNA), preventing DNA strand invasion and exchange by homologous recombination. This activity requires a physical interaction between Srs2 and Rad51, which stimulates ATP turnover in the Rad51 nucleoprotein filament and causes dissociation of Rad51 from ssDNA. Srs2 also possesses a DNA unwinding activity and here we show that assembly of more than one Srs2 molecule on the 3′ ssDNA overhang is required to initiate DNA unwinding. When Rad51 is bound on the double-stranded DNA, its interaction with Srs2 blocks the helicase (DNA unwinding) activity of Srs2. Thus, in different DNA contexts, the physical interaction of Rad51 with Srs2 can either stimulate or inhibit the remodeling functions of Srs2, providing a means for tailoring DNA strand exchange activities to enhance the fidelity of recombination

Synthetic Lethality of Chk1 Inhibition Combined with p53 and/or p21 Loss During a DNA Damage Response in Normal and Tumor Cells

Cell cycle checkpoints ensure genome integrity and are frequently compromised in human cancers. A therapeutic strategy being explored takes advantage of checkpoint defects in p53-deficient tumors in order to sensitize them to DNA-damaging agents by eliminating Chk1-mediated checkpoint responses. Using mouse models, we demonstrated that p21 is a key determinant of how cells respond to the combination of DNA damage and Chk1 inhibition (combination therapy) in normal cells as well as in tumors. Loss of p21 sensitized normal cells to the combination therapy much more than did p53 loss and the enhanced lethality was partially blocked by CDK inhibition. In addition, basal pools of p21 (p53 independent) provided p53 null cells with protection from the combination therapy. Our results uncover a novel p53-independent function for p21 in protecting cells from the lethal effects of DNA damage followed by Chk1 inhibition. As p21 levels are low in a significant fraction of colorectal tumors, they are predicted to be particularly sensitive to the combination therapy. Results reported in this study support this prediction

Contributions Made by CDC25 Phosphatases to Proliferation of Intestinal Epithelial Stem and Progenitor Cells

The CDC25 protein phosphatases drive cell cycle advancement by activating cyclin-dependent protein kinases (CDKs). Humans and mice encode three family members denoted CDC25A, -B and -C and genes encoding these family members can be disrupted individually with minimal phenotypic consequences in adult mice. However, adult mice globally deleted for all three phosphatases die within one week after Cdc25 disruption. A severe loss of absorptive villi due to a failure of crypt epithelial cells to proliferate was observed in the small intestines of these mice. Because the Cdc25s were globally deleted, the small intestinal phenotype and loss of animal viability could not be solely attributed to an intrinsic defect in the inability of small intestinal stem and progenitor cells to divide. Here, we report the consequences of deleting different combinations of Cdc25s specifically in intestinal epithelial cells. The phenotypes arising in these mice were then compared with those arising in mice globally deleted for the Cdc25s and in mice treated with irinotecan, a chemotherapeutic agent commonly used to treat colorectal cancer. We report that the phenotypes arising in mice globally deleted for the Cdc25s are due to the failure of small intestinal stem and progenitor cells to proliferate and that blocking cell division by inhibiting the cell cycle engine (through Cdc25 loss) versus by inducing DNA damage (via irinotecan) provokes a markedly different response of small intestinal epithelial cells. Finally, we demonstrate that CDC25A and CDC25B but not CDC25C compensate for each other to maintain the proliferative capacity of intestinal epithelial stem and progenitor cells

Type I Interferons Link Viral Infection to Enhanced Epithelial Turnover and Repair

The host immune system functions constantly to maintain chronic commensal and pathogenic organisms in check. The consequences of these immune responses on host physiology are as yet unexplored, and may have long-term implications in health and disease. We show that chronic viral infection increases epithelial turnover in multiple tissues, and the antiviral cytokines type I interferons (IFNs) mediate this response. Using a murine model with persistently elevated type I IFNs in the absence of exogenous viral infection, the Irgm1−/− mouse, we demonstrate that type I IFNs act through nonepithelial cells, including macrophages, to promote increased epithelial turnover and wound repair. Downstream of type I IFN signaling, the highly related IFN-stimulated genes Apolipoprotein L9a and b activate epithelial proliferation through ERK activation. Our findings demonstrate that the host immune response to chronic viral infection has systemic effects on epithelial turnover through a myeloid-epithelial circuit

Loss of homeostasis in small intestines of <i>vABKO a</i>nd <i>vTKO</i> mice.

<p>(<b>A</b>) Mice were injected with tamoxifen for five consecutive days and then sacrificed 3 days after the final injection. Small intestines were isolated and length determinations were made. Small intestine lengths were normalized to body weights, which were determined prior to the first tamoxifen-injection. Data is presented as mean +/− SEM. Asterisk (*) indicates significantly different after tamoxifen injection as determined by a Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. The actual P-values are 0.38 (WT), 0.76 (<i>vil-Cre-ER^T2</i>), 0.003 (<i>vAKO</i>), 0.31 (<i>vBKO</i>), 0.002 (<i>vABKO</i>), 0.46 (<i>vACKO</i>) and 0.004 (<i>vTKO</i>). The small intestinal lengths of <i>vAKO</i>, <i>vABKO</i> and <i>vTKO</i> mice were significantly different from WT mice injected with tamoxifen as determined by a Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. Actual P-values are 0.40 (<i>vil-Cre-ER^T2</i>), 0.03 (<i>vAKO</i>), 0.10 (<i>vBKO</i>), 0.00006 (<i>vABKO</i>), 0.36 (<i>vACKO</i>) and 0.0006 (<i>vTKO</i>). (<b>B</b>) Duodenums isolated from mice treated as described in A were photographed under a dissection microscope. Scale bar: 0.5 mm. (<b>C</b>) Significant shortening of villi in small intestines of <i>vABKO</i> and <i>vTKO</i> mice. Length of individual villi shown in panel B were measured (30 villi per mouse). Data is presented as mean +/− SEM. Asterisk (*) indicates significantly different after tamoxifen injection as determined by a Student's t-test. P-values are 0.00008 (<i>vABKO</i>) and 0.007 (<i>vTKO</i>). Villi lengths of <i>vil-Cre-ER^T2</i>, <i>vABKO</i> and <i>vTKO</i> mice were significantly different from WT mice injected with tamoxifen as determined by a Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. Actual P-values are 0.001 (<i>vil-Cre-ER^T2</i>), 0.27 (<i>vAKO</i>), 0.45 (<i>vBKO</i>), 0.0005 (<i>vABKO</i>), 0.25 (<i>ACKO</i>) and 0.03 (<i>vTKO</i>).</p

Tamoxifen injection induced efficient recombination within <i>Cdc25A</i> and <i>Cdc25B</i> loci.

<p>(<b>A</b>) Genomic DNA isolated from the small and large intestines of tamoxifen-treated mice were assessed for Cre-mediated excision by Southern blotting. Deletion frequencies are shown below each lane and were determined by measuring band intensities using a Molecular Dynamics Storm imager. (B) Total RNA isolated from the small intestine (jejunum) of tamoxifen-treated WT and <i>vB^f/−</i> mice was reverse-transcribed into cDNA. qRT-PCR was used to determine relative amounts of <i>Cdc25B</i> mRNA. The data is presented as mean +/− SEM. Asterisk (*) indicates significantly different from WT, P  =  0.012 by Student's t-test. Note that <i>vB^f/−</i> mice are generated by crossing <i>Cdc25B</i> null mice <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015561#pone.0015561-Lincoln1" target="_blank">[26]</a> with <i>Cdc25B</i> conditional mice. The PCR primers detect transcript arising from the null allele but not the deleted floxed allele. Thus, a 50% decrease in relative expression indicates complete loss of <i>Cdc25B</i> expression.</p

Proliferation and apoptosis in crypts of <i>Cdc25</i>-disrupted mice.

<p>(<b>A</b>) Mice were injected with tamoxifen for five consecutive days and then sacrificed 3 days after the final injection. One hour prior to sacrifice, mice were injected with BrdU. Intestines were isolated, and sections were stained with BrdU antibody (brown) and counterstained with hematoxylin (blue). Scale bar: 20 mm. (<b>B</b>) Mice were injected with tamoxifen for five consecutive days and were sacrificed 3 days after last tamoxifen injection. Intestines were isolated and sections were stained for cleaved caspase-3. 3, 3′-diaminobenzidine (DAB, brown) was used as a substrate, and sections were counter-stained with hematoxylin. Arrows indicate cells at the tip of villi, which stain positive for cleaved caspase-3. Scale bar: 0.1 mm. (<b>C–D</b>) Crypts within the proximal portion of the small intestine of tamoxifen treated mice were examined for mitotic cells (presence of mitotic figures) (C) and apoptotic cells (presence of fragmented nuclei) (D). Areas containing Brunner's gland were excluded from analysis. Twenty crypts were counted per mouse and three mice of each genotype were evaluated. Similar patterns were observed in mid and distal portions of the small intestine (data not shown). Data is presented as mean +/− SEM. Asterisks in panel C indicate significantly different from WT mice as determined by a Student's t-test (M-phase cells). *, P<0.05; **, P<0.01; ***, P<0.001. Actual P-values are 0.22 (<i>vil-Cre-ER^T2</i>), 0.045 (<i>vAKO</i>), 0.16 (<i>vBKO</i>), 0.00056 (<i>vABKO</i>), 0.17 (<i>ACKO</i>) and 0.00055 (<i>vTKO</i>). Asterisks in panel D indicate significantly different number of apoptotic cells from <i>WT</i> mice as determined by a Student's t-test. P-values are 0.48 (<i>vAKO</i>), 0.33 (<i>vBKO</i>), 0.0011 (<i>vABKO</i>), 0.064 (<i>ACKO</i>) and 0.0010 (<i>vTKO</i>). (<b>E</b>) Mice were injected with tamoxifen as described in A and small intestines were isolated, sectioned and stained for inactive Cdk1 (phosphorylated on Tyr-15, brown) and counterstained with hematoxylin (blue). Arrows indicate epithelial cells positively stained with the phospho-Tyr 15 CDK1 antibody. Scale bar: 20 µm.</p