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

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

Response of crypt epithelial cells to loss of CDC25s.

<p>(<b>A</b>) Mice were treated with tamoxifen for five consecutive days and sacrificed 3 days after the final injection. Small intestines were isolated and fixed. 5 µm tissue sections were prepared and stained with Hematoxylin and Eosin. Arrow heads indicate Paneth cells. Scale bar: 0.5 mm. (<b>B</b>) Crypts within the proximal portion of the small intestine of tamoxifen treated mice were examined for epithelial cells and Paneth cells. 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. Asterisk (*) indicates significantly different from WT mice as determined by a Student's t-test (total epithelial cells). *, P<0.05; **, P<0.01; ***, P<0.001. Actual P-values are 0.41 (<i>vil-Cre-ER^T2</i>), 0.21 (<i>vAKO</i>), 0.23 (<i>vBKO</i>), 0.002 (<i>vABKO</i>), 0.15 (<i>ACKO</i>) and 0.0007 (<i>vTKO</i>). <i>vBKO</i> mice have slightly more mature Paneth cells per crypt compared to WT mice as determined by a Student's t-test. P-values are 0.52 (<i>vil-Cre-ER^T2</i>), 0.77 (<i>vAKO</i>), 0.044 (<i>vBKO</i>), 0.28 (<i>vABKO</i>), 0.076 (<i>ACKO</i>) and 0.098 (<i>vTKO</i>).</p

Specific deletion of <i>Cdc25A</i> in intestinal epithelial cells.

<p>(<b>A</b>, <b>B</b>) <i>vR26R</i>;<i>A^f/−</i> and v<i>A^f/−</i> mice were injected with tamoxifen for 5 consecutive days and then sacrificed 3 days after the final injection. Frozen sections of small intestines were prepared and stained with X-gal to visualize Cre-mediated deletion frequencies. Scale bar: 200 µm. (<b>C</b>) Genomic DNA isolated from the small and large intestines of <i>vA^f/−</i> mice 3 days after the final tamoxifen-injection was digested with Bst XI followed by Southern blotting. Deletion frequencies are shown below the each lane. (<b>D</b>) Mice were weighed prior to injection and 3 days after the final tamoxifen injection. Data is presented as mean +/− standard error of the mean (SEM). Asterisks indicate 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. The actual P-values are 0.86 (<i>vil-Cre-ER^T2</i>), 0.15 (<i>vAKO</i>), 0.87 (<i>vBKO</i>), 0.00007 (<i>vABKO</i>), 0.11(<i>vACKO</i>) and 0.0004 (<i>vTKO</i>).</p