_:b439699699 . _:b439699700 . _:b439699701 . _:b439699702 . _:b439699703 . _:b439699688 . _:b439699689 . _:b439699690 . _:b8381223 "are often associated with spontaneous genotoxic stress, and the resulting DNA damage signaling can elicit tumor-suppressive responses such as cell senescence or apoptosis (Bartkova et al., 2006; Di Micco et al., 2006; Toledo et al., >>2008<<). Mutations that dampen the cellular response to DNA damage signals, such as loss of p53, or attenuate the initial generation of such signals, as we propose in this study for Akt, would be subject to powerful selection by allowing cells" . _:b439699691 . _:b439699692 . _:b439699693 . _:b439699694 . _:b439699695 . _:b439699653 . _:b439699680 . _:b439699681 . _:b439699682 . _:b439699683 . _:b439699703 . _:b439699637 "5"^^ . _:b439699684 . _:b439699685 . _:b439699686 . . _:b439699687 . _:b439699672 . . _:b439699673 . _:b439699636 "6"^^ . . _:b439699674 . _:b439699675 . _:b439699639 "4"^^ . _:b439699723 . _:b439699676 . _:b439699677 . _:b439699678 . _:b439699679 . _:b439699664 . _:b439699638 "5"^^ . _:b439699665 . _:b439699666 . _:b439699634 "7"^^ . . _:b439699667 . _:b439699668 . _:b439699669 . _:b439699669 . _:b8381183 . _:b8381168 . _:b439699635 "7"^^ . _:b439699670 . _:b439699671 . _:b8381213 . _:b439699656 . _:b439699657 . _:b439699658 . _:b439699659 . _:b439699660 . _:b439699661 . _:b439699662 . _:b439699711 . _:b439699663 . _:b439699648 . . _:b439699649 . _:b439699650 . _:b439699645 "3"^^ . _:b439699651 . _:b439699652 . _:b439699653 . _:b439699637 . _:b439699644 "3"^^ . _:b439699654 . . _:b439699655 . _:b439699640 . _:b439699641 . _:b439699642 . _:b439699647 "3"^^ . _:b8381188 "TopBP1-ER function was activated by adding tamoxifen to the culture medium as described previously (Toledo et al., >>2008<<). In general, cells were treated with tamoxifen for 2 h before harvesting. To augment Akt activity, HCT116 cells were transiently transfected with a plasmid encoding a constitutively active gag-Akt fusion protein (Burgering and Coffer," . _:b439699643 . _:b439699644 . _:b439699645 . . _:b439699646 "3"^^ . . _:b439699646 . _:b8381195 "To assess checkpoint proficiency in late G2, we used a DT40 cell line, Cdk1AS, in which a mutant, analogue-sensitive (AS) form of Cdk1 replaces the endogenous kinase (Hochegger et al., >>2007<<). When exposed to the ATP analogue 1NM-PP1, Cdk1AS cells accumulated homogenously in G2, and when the drug was washed away, the great majority rapidly entered mitosis and divided (Fig. 1 A; Hochegger et al., 2007). Importantly, the" . _:b439699712 . _:b439699647 . _:b439699641 "3"^^ . _:b439699634 . _:b8381210 . _:b439699635 . _:b439699691 . _:b439699636 . _:b439699637 . _:b439699638 . _:b439699640 "4"^^ . _:b439699639 . _:b439699643 "3"^^ . _:b8381183 "materials and methods" . _:b439699642 "3"^^ . _:b439699653 "3"^^ . _:b8381206 . _:b439699672 . _:b439699674 . _:b439699652 "3"^^ . _:b439699655 "2"^^ . _:b8381209 . _:b439699654 "2"^^ . . _:b439699696 . _:b439699649 "3"^^ . _:b8381192 . . _:b8381193 "results and discussion" . _:b439699648 "3"^^ . _:b439699651 "3"^^ . . _:b439699650 "3"^^ . _:b8381196 "When exposed to the ATP analogue 1NM-PP1, Cdk1AS cells accumulated homogenously in G2, and when the drug was washed away, the great majority rapidly entered mitosis and divided (Fig. 1 A; Hochegger et al., >>2007<<). Importantly, the negative regulatory phosphorylation on tyrosine 15 (Y15), which restrains Cdk1 catalytic activity before mitosis and forms the principal target of the DNA damage checkpoint, is maintained in 1NM-PP1\u2013arrested cells" . _:b8381193 . _:b439699727 . _:b439699661 "2"^^ . _:b439699660 "2"^^ . _:b439699663 "2"^^ . _:b439699662 "2"^^ . _:b439699693 . _:b439699657 "2"^^ . _:b439699655 . _:b8381198 "These observations indicate that ATM is activated by DNA damage in late G2 cells; however, irradiation-induced mitotic delay in DT40 cells has been shown to be dependent on ATR-mediated activation of Chk1 (Zachos et al., >>2003<<; Walker et al., 2009)." . _:b439699656 "2"^^ . _:b439699659 "2"^^ . _:b8381194 . _:b439699658 "2"^^ . _:b439699669 "2"^^ . . . _:b439699668 "2"^^ . . _:b439699671 "2"^^ . _:b439699670 "2"^^ . . . _:b439699665 "2"^^ . _:b439699664 "2"^^ . _:b439699718 . _:b439699667 "2"^^ . _:b439699666 "2"^^ . _:b439699677 "2"^^ . _:b439699676 "2"^^ . _:b8381190 "For ATR and RPA immunocytochemistry, cells were pretreated with extraction buffer (25 mM Hepes, pH 7.4, 50 mM NaCl, 1 mM ETDA, 3 mM MgCl2, 300 mM sucrose, and 0.5% Triton X-100) as described previously (Huertas and Jackson, >>2009<<) before fixation and permeabilization as for Chk1 pS345, \u03B3-H2AX, and Rad51. For CtIP immunocytochemistry, cells were fixed with 4% paraformaldehyde for 15 min and then treated with 70% EtOH in PBS at \u221220\u00B0C." . _:b439699679 "2"^^ . _:b8381178 "Phosphorylation of Chk1 by ATR at serine 345 (S345) within the C-terminal regulatory domain in particular is essential for both the DNA damage and replication checkpoint responses in vertebrates (Walker et al., >>2009<<)." . _:b439699678 "2"^^ . _:b439699671 . _:b439699673 "2"^^ . _:b439699672 "2"^^ . . _:b439699648 . _:b439699675 "2"^^ . _:b439699674 "2"^^ . . _:b8381204 . _:b439699709 . _:b439699685 "2"^^ . . _:b439699684 "2"^^ . . _:b439699687 "2"^^ . . _:b439699686 "2"^^ . _:b439699681 "2"^^ . _:b439699680 . _:b8381215 . _:b8381203 "1NM-PP1\u2013arrested Cdk1AS cells express high levels of Cdk2 activity (Hochegger et al., >>2007<<; Bourke et al., 2010), which is known to be sufficient to sustain both resection and checkpoint activation (Cerqueira et al., 2009)." . _:b439699680 "2"^^ . . _:b439699683 "2"^^ . _:b439699682 "2"^^ . _:b8381222 "stages of neoplasia are often associated with spontaneous genotoxic stress, and the resulting DNA damage signaling can elicit tumor-suppressive responses such as cell senescence or apoptosis (Bartkova et al., 2006; Di Micco et al., >>2006<<; Toledo et al., 2008)." . _:b439699693 "2"^^ . _:b439699692 "2"^^ . _:b8381188 . _:b8381180 "Consistent with this, Chk1 was activated most strongly in fractions enriched for S- and G2-phase cells when irradiated DT40 cell cultures were fractionated by elutriation (Walker et al., >>2009<<). Cell cycle\u2013dependent DSB processing to generate single-stranded DNA is likely to play a role in determining this pattern of ATR\u2013Chk1 activation (Jazayeri et al., 2006); however, it remains possible that other cell cycle phase\u2013specific" . _:b439699695 "2"^^ . _:b8381189 . _:b439699717 . . _:b439699694 "2"^^ . _:b439699673 . _:b8381185 . _:b8381190 . _:b8381208 "Because Akt/PKB kinase activity has been reported to override DNA damage\u2013induced G2 arrest (Henry et al., 2001; Shtivelman et al., >>2002<<; Nimbalkar and Quelle, 2008), we examined the effect of two selective Akt inhibitors, Akt-I and Akt1/2-I." . _:b439699716 . . _:b439699689 "2"^^ . . . _:b8381191 . _:b439699719 . . _:b439699688 "2"^^ . _:b439699652 . _:b8381184 . . _:b439699718 . _:b439699704 . . _:b439699691 "2"^^ . _:b8381185 . _:b439699713 . _:b439699690 "2"^^ . _:b8381186 . _:b439699712 . _:b439699701 "2"^^ . _:b8381187 . _:b439699714 . _:b439699715 . _:b439699700 "2"^^ . _:b8381196 . _:b439699714 . _:b439699703 "2"^^ . _:b8381197 . _:b439699725 . _:b439699702 "2"^^ . _:b8381198 . _:b439699724 . _:b439699646 . _:b439699697 "2"^^ . _:b8381199 . _:b439699638 . _:b439699727 . _:b439699696 "2"^^ . _:b8381199 . _:b8381192 . _:b439699726 . _:b439699699 "2"^^ . . . _:b8381193 . _:b439699721 . _:b439699698 "2"^^ . . _:b8381194 . _:b439699667 . _:b439699720 . _:b439699709 "2"^^ . _:b8381195 . _:b439699723 . _:b8381220 . _:b439699708 "2"^^ . _:b8381204 . . _:b439699722 . _:b439699711 "2"^^ . . _:b8381205 . _:b439699710 "2"^^ . _:b8381206 . . _:b439699705 "2"^^ . _:b8381207 . . _:b439699704 "2"^^ . _:b8381200 . _:b439699707 "2"^^ . _:b8381201 . _:b439699729 . _:b439699706 "2"^^ . _:b8381170 "In part, this is controlled through Cdk-mediated phosphorylation of Sae2, a protein required to initiate the resection process (Huertas et al., >>2008<<). Vertebrate cells express an orthologue of Sae2, CtIP (C-terminal interacting protein), which is also crucial for DSB resection (Sartori et al., 2007). Strand resection is also cell cycle regulated in vertebrate cells, and evidence" . _:b8381202 . _:b439699728 . . _:b439699717 "2"^^ . _:b8381203 . _:b439699658 . _:b439699716 "2"^^ . . . _:b8381212 . _:b8381170 . _:b439699719 "2"^^ . _:b8381213 . _:b439699718 "2"^^ . _:b8381214 . _:b439699713 "2"^^ . _:b8381215 . _:b8381187 "Fernandez-Capetillo, Spanish National Cancer Research Centre, Madrid, Spain; Toledo et al., >>2008<<) and selected for resistance to puromycin." . _:b439699712 "2"^^ . _:b8381221 "The early stages of neoplasia are often associated with spontaneous genotoxic stress, and the resulting DNA damage signaling can elicit tumor-suppressive responses such as cell senescence or apoptosis (Bartkova et al., >>2006<<; Di Micco et al., 2006; Toledo et al., 2008)." . _:b8381208 . _:b439699700 . _:b439699715 "2"^^ . . _:b8381209 . _:b8381206 "Plk1 kinase is active in G2 and is required for recovery from DNA damage\u2013induced checkpoint arrest (Mac\u016Frek et al., >>2008<<); however, pretreatment with Plk1 inhibitor did not restore Chk1 activation after irradiation in late G2\u2013arrested cells (Fig." . _:b439699714 "2"^^ . _:b8381210 . . _:b439699725 "2"^^ . _:b439699697 . _:b8381211 . . _:b439699724 "2"^^ . _:b8381220 . . _:b439699727 "2"^^ . . _:b8381221 . _:b439699657 . . _:b439699677 . . . _:b439699726 "2"^^ . _:b439699683 . _:b8381222 . _:b439699656 . _:b439699721 "2"^^ . _:b8381176 "simply through association with DSBs (Harrison and Haber, 2006), ATR is activated through recruitment to regions of single-stranded DNA in association with its partner protein, ATRIP (ATR-interacting protein; Cimprich and Cortez, >>2008<<). Once activated, ATR and ATM selectively phosphorylate and activate two downstream checkpoint effector kinases, Chk1 and Chk2 (Harrison and Haber, 2006)." . . _:b8381223 . _:b439699720 "2"^^ . _:b8381216 . _:b439699723 "2"^^ . _:b8381217 . _:b439699722 "2"^^ . _:b8381218 . . _:b8381168 _:b8381172 . _:b8381168 _:b8381173 . _:b8381168 _:b8381174 . _:b8381219 . _:b8381168 _:b8381175 . _:b8381199 "indicate that ATM is activated by DNA damage in late G2 cells; however, irradiation-induced mitotic delay in DT40 cells has been shown to be dependent on ATR-mediated activation of Chk1 (Zachos et al., 2003; Walker et al., >>2009<<). Strikingly, although Chk1 S345 phosphorylation could be induced by irradiation of asynchronous cultures, no corresponding activation occurred in G2-arrested cells (Fig." . _:b8381168 _:b8381169 . _:b8381168 _:b8381170 . _:b8381168 _:b8381171 . _:b8381217 . _:b8381168 _:b8381180 . _:b8381168 _:b8381181 . _:b8381168 _:b8381182 . _:b8381168 _:b8381176 . _:b8381168 _:b8381177 . _:b8381168 _:b8381178 . _:b8381168 _:b8381179 . _:b439699729 "2"^^ . . . . _:b439699728 "2"^^ . _:b8381186 . _:b8381175 . . _:b8381177 . . . _:b439699670 . _:b8381189 . _:b439699675 . . . . _:b8381172 . _:b439699726 . . . _:b439699659 . _:b439699728 . _:b8381182 "Finally, it has been reported that Chk1 becomes refractory to activation by DNA damage in mitotic cells (Shiromizu et al., >>2006<<); however, when this desensitization occurs and whether it is imposed via the same regulatory processes that operate during interphase are unknown." . _:b439699729 . . _:b439699707 . _:b439699720 . _:b439699721 . _:b439699722 . . _:b8381209 "Because Akt/PKB kinase activity has been reported to override DNA damage\u2013induced G2 arrest (Henry et al., 2001; Shtivelman et al., 2002; Nimbalkar and Quelle, >>2008<<), we examined the effect of two selective Akt inhibitors, Akt-I and Akt1/2-I." . _:b439699728 . _:b439699723 . _:b439699724 . _:b8381215 "We postulate that Akt inhibits the expression or activity of one or more factors required for DSB resection, perhaps opposing the positive effects of Cdk-mediated phosphorylation (Wohlbold and Fisher, >>2009<<). Interestingly, Akt activity is strongly cell cycle regulated in Cdk1AS cells with very high levels in G1, low levels in S, and intermediate levels again in G2 (Fig. S1 A and not depicted). The basis of this regulation is not understood;" . _:b439699725 . _:b439699726 . _:b439699727 . _:b439699719 . _:b439699712 . _:b439699713 . _:b439699714 . _:b8381200 . _:b439699715 . _:b439699716 . _:b439699717 . _:b439699718 . _:b439699719 . . _:b439699721 . . . _:b8381216 "The basis of this regulation is not understood; however, similar fluctuations have been observed in other cell types in which Akt activity has also been implicated in regulation of the G2/M transition (Shtivelman et al., >>2002<<). Interestingly, the level of Mre11, a factor required for strand resection, was markedly reduced in PTEN\u2212/\u2212 HCT116 cells compared with controls (Fig. 5 A), whereas constitutive Akt activity has been reported to inhibit recombination by" . _:b439699651 . _:b8381179 "Thus, in human T24 cultures released from density arrest, Chk1 was activated in response to irradiation only in cells that had reached S and G2 phase (Jazayeri et al., >>2006<<). Consistent with this, Chk1 was activated most strongly in fractions enriched for S- and G2-phase cells when irradiated DT40 cell cultures were fractionated by elutriation (Walker et al., 2009). Cell cycle\u2013dependent DSB processing to" . _:b439699722 . _:b8381184 "Waldman, Lombardi Comprehensive Cancer Center, Georgetown University School of Medicine, Washington, DC; Lee et al., >>2004<<) were grown in DME containing 10% fetal bovine serum at 37\u00B0C in 5% CO2." . _:b8381220 "It has generally been considered that Akt deregulation either drives tumor cell proliferation directly or enhances cell survival (Manning and Cantley, >>2007<<). Our findings suggest an alternative, complementary possibility, namely that Akt activation may allow cells to evade the deleterious consequences of DNA damage. The early stages of neoplasia are often associated with spontaneous" . . _:b439699717 . "PMC0" . . _:b439699686 . _:b8381192 "For S-phase determinations, cells were labeled with 25 \u00B5M BrdU and then fixed and stained using anti-BrdU monoclonal antibody as described previously (Robinson et al., >>2006<<)." . . _:b8381172 "Strand resection is also cell cycle regulated in vertebrate cells, and evidence suggests that Cdks may regulate this process at least in part via direct phosphorylation of CtIP in a manner analogous to yeast (Huertas and Jackson, >>2009<<; Yun and Hiom, 2009). However, whether this is the only mechanism underlying cell cycle regulation of DSB resection in vertebrates is unclear." . . _:b8381212 . . _:b439699644 . _:b439699663 . . _:b439699636 . . . _:b8381178 . . _:b439699724 . . _:b8381175 "Unlike the related PIKK ATM (ataxia telangiectasia mutated), which can be activated simply through association with DSBs (Harrison and Haber, >>2006<<), ATR is activated through recruitment to regions of single-stranded DNA in association with its partner protein, ATRIP (ATR-interacting protein; Cimprich and Cortez, 2008)." . . _:b439699695 . _:b439699713 . _:b8381211 "Oncogenic activation of Akt frequently results from loss of PTEN expression or function (Chalhoub and Baker, >>2009<<). To determine how PTEN loss affects DNA damage signaling, we took advantage of a PTEN-deficient HCT116 derivative (Lee et al., 2004)." . . _:b8381180 . . _:b439699641 . _:b8381217 "5 A), whereas constitutive Akt activity has been reported to inhibit recombination by inducing cytoplasmic sequestration of Rad51 and Brca1 (Plo et al., >>2008<<). Although suggestive of possible mechanisms, further work will be required to identify the target or targets through which Akt modulates DNA damage signaling." . _:b8381223 . _:b439699645 . _:b8381200 "We next used the reversible Cdk1-selective inhibitor, RO-3306 (Vassilev et al., >>2006<<), to arrest HCT116 cells in late G2 phase and then released them with or without prior irradiation." . _:b8381184 . _:b439699694 . . _:b8381211 . _:b8381181 "Cell cycle\u2013dependent DSB processing to generate single-stranded DNA is likely to play a role in determining this pattern of ATR\u2013Chk1 activation (Jazayeri et al., >>2006<<); however, it remains possible that other cell cycle phase\u2013specific processes could also contribute." . . . _:b8381219 "Akt is deregulated in many human tumors (Yuan and Cantley, >>2008<<). It has generally been considered that Akt deregulation either drives tumor cell proliferation directly or enhances cell survival (Manning and Cantley, 2007)." . . _:b439699689 . _:b8381176 . . . _:b8381174 . . _:b439699725 . _:b8381218 "This is unlikely to be mediated via direct phosphorylation of Chk1 by Akt (Puc et al., >>2005<<) because a Chk1 S280A mutant is similarly affected (Fig." . . _:b8381195 . _:b439699710 . . _:b8381197 "negative regulatory phosphorylation on tyrosine 15 (Y15), which restrains Cdk1 catalytic activity before mitosis and forms the principal target of the DNA damage checkpoint, is maintained in 1NM-PP1\u2013arrested cells (Hochegger et al., >>2007<<)." . . _:b439699690 . _:b8381213 "Interestingly, a recent study demonstrated a link between the dissolution of Rad51 foci and mitotic entry (Ayoub et al., >>2009<<). Therefore, one possibility is that DNA strand resection may be switched off late in G2 simply to terminate recombination in preparation for mitotic entry and chromosome segregation. Because recombination and ATR activation are both" . . _:b8381171 . _:b439699708 . . _:b439699716 . _:b8381196 . . _:b8381197 . _:b8381182 . _:b8381218 . _:b439699664 . _:b439699662 . _:b8381187 . . _:b439699679 . . _:b8381189 "To augment Akt activity, HCT116 cells were transiently transfected with a plasmid encoding a constitutively active gag-Akt fusion protein (Burgering and Coffer, >>1995<<). To inhibit Plk1, Akt, and ATM, cells were pretreated for 1 h with 0.1 \u00B5M Plk1 inhibitor (BI 2536), 25 \u00B5M Akt1/2-I inhibitor (Sigma-Aldrich), 5 \u00B5M Akt-I (EMD), or 10 \u00B5M ATM inhibitor Ku55933 (Tocris Bioscience) before experimental" . _:b439699637 . _:b8381188 . _:b439699636 . . _:b8381207 "Because Akt/PKB kinase activity has been reported to override DNA damage\u2013induced G2 arrest (Henry et al., >>2001<<; Shtivelman et al., 2002; Nimbalkar and Quelle, 2008), we examined the effect of two selective Akt inhibitors, Akt-I and Akt1/2-I." . _:b439699639 . . _:b439699660 . _:b439699638 . . . _:b8381207 . _:b439699635 . _:b439699668 . _:b439699634 . . _:b8381177 "Once activated, ATR and ATM selectively phosphorylate and activate two downstream checkpoint effector kinases, Chk1 and Chk2 (Harrison and Haber, >>2006<<). Phosphorylation of Chk1 by ATR at serine 345 (S345) within the C-terminal regulatory domain in particular is essential for both the DNA damage and replication checkpoint responses in vertebrates (Walker et al., 2009)." . . _:b439699645 . _:b8381202 "To confirm that the ATR\u2013Chk1 pathway was potentially functional in late G2\u2013arrested cells, we expressed a TopBP1-ER fusion protein that activates ATR in response to tamoxifen (Toledo et al., >>2008<<). Tamoxifen activated Chk1 as potently as irradiation when added to proliferating Cdk1AS\u2013TopBP1-ER cells (Fig." . _:b8381203 . . _:b8381204 "1NM-PP1\u2013arrested Cdk1AS cells express high levels of Cdk2 activity (Hochegger et al., 2007; Bourke et al., >>2010<<), which is known to be sufficient to sustain both resection and checkpoint activation (Cerqueira et al., 2009)." . _:b439699644 . _:b439699647 . _:b8381202 . _:b439699646 . _:b439699641 . _:b8381212 "To determine how PTEN loss affects DNA damage signaling, we took advantage of a PTEN-deficient HCT116 derivative (Lee et al., >>2004<<). PTEN\u2212/\u2212 HCT116 cells exhibited much higher levels of active S473-phosphorylated Akt than either parental HCT116 cells or a drug-resistant control (HCT116NeoR; Fig. 5 A). Strikingly, activation of Chk1 S345 phosphorylation after" . _:b8381205 . _:b439699640 . _:b439699643 . _:b439699682 . _:b439699681 . _:b439699642 . _:b439699653 . . _:b439699652 . _:b8381205 "1NM-PP1\u2013arrested Cdk1AS cells express high levels of Cdk2 activity (Hochegger et al., 2007; Bourke et al., 2010), which is known to be sufficient to sustain both resection and checkpoint activation (Cerqueira et al., >>2009<<). Therefore, we considered that some other pathway or kinase active in G2 might actively suppress DNA damage processing." . _:b439699655 . . _:b439699654 . _:b439699649 . _:b8381185 "separated in an elutriating rotor (JE-6B; Beckman Coulter) at room temperature at a flow rate of 40 ml/min, and fractions enriched for G1/early S (F1) and late S/G2/M (F2) cells were isolated as described previously (Walker et al., >>2009<<). Cells were irradiated using an Alcyon II CGR MeV cobalt source as described previously (Rainey et al., 2008)." . _:b439699687 . _:b439699648 . _:b439699661 . _:b439699651 . _:b8381173 "is also cell cycle regulated in vertebrate cells, and evidence suggests that Cdks may regulate this process at least in part via direct phosphorylation of CtIP in a manner analogous to yeast (Huertas and Jackson, 2009; Yun and Hiom, >>2009<<). However, whether this is the only mechanism underlying cell cycle regulation of DSB resection in vertebrates is unclear." . . _:b439699650 . _:b439699684 . _:b439699715 . _:b439699661 . _:b439699660 . _:b439699663 . _:b8381193 _:b8381194 . . _:b439699662 . . _:b8381193 _:b8381195 . _:b8381169 "In yeasts, Cdk activity plays a major role in controlling DNA strand resection (Wohlbold and Fisher, >>2009<<). In part, this is controlled through Cdk-mediated phosphorylation of Sae2, a protein required to initiate the resection process (Huertas et al., 2008). Vertebrate cells express an orthologue of Sae2, CtIP (C-terminal interacting" . _:b8381193 _:b8381196 . _:b8381193 _:b8381197 . _:b8381193 _:b8381198 . _:b439699657 . _:b8381193 _:b8381199 . _:b8381168 "introduction" . _:b8381193 _:b8381200 . _:b8381193 _:b8381201 . _:b8381193 _:b8381202 . _:b439699656 . _:b8381193 _:b8381203 . _:b8381193 _:b8381204 . _:b8381193 _:b8381205 . _:b439699665 . _:b8381193 _:b8381206 . _:b439699659 . _:b8381193 _:b8381207 . _:b8381193 _:b8381208 . _:b8381193 _:b8381209 . _:b8381193 _:b8381210 . _:b439699658 . _:b8381193 _:b8381211 . _:b8381193 _:b8381212 . _:b8381201 "Because CtIP is required for DNA strand resection at DSBs (Sartori et al., >>2007<<), it seems likely that failure to recruit this component to sites of damage contributes to the resection defect." . _:b8381193 _:b8381213 . . _:b8381193 _:b8381214 . . _:b439699669 . _:b8381193 _:b8381215 . _:b8381193 _:b8381216 . _:b8381193 _:b8381217 . _:b8381193 _:b8381218 . _:b439699668 . _:b8381193 _:b8381219 . _:b8381193 _:b8381220 . _:b8381174 "tracts of single-stranded DNA play a key role in triggering aspects of the DNA damage checkpoint response by recruiting and activating the PIKK (PI3-kinase\u2013like kinase) ATR (ataxia telangiectasia and Rad3 related; Cimprich and Cortez, >>2008<<). Unlike the related PIKK ATM (ataxia telangiectasia mutated), which can be activated simply through association with DSBs (Harrison and Haber, 2006), ATR is activated through recruitment to regions of single-stranded DNA in association" . _:b8381193 _:b8381221 . _:b8381193 _:b8381222 . _:b439699671 . _:b8381193 _:b8381223 . _:b8381171 "Vertebrate cells express an orthologue of Sae2, CtIP (C-terminal interacting protein), which is also crucial for DSB resection (Sartori et al., >>2007<<). Strand resection is also cell cycle regulated in vertebrate cells, and evidence suggests that Cdks may regulate this process at least in part via direct phosphorylation of CtIP in a manner analogous to yeast (Huertas and Jackson, 2009;" . . _:b439699670 . . . _:b439699665 . . _:b439699664 . . _:b439699667 . _:b8381214 "However, checkpoint deactivation might also be required for a normal G2/M transition because recent data have shown that Chk1 and Cdk1 interact in a feedback loop that contributes to timing mitotic entry (Enomoto et al., >>2009<<)." . _:b439699666 . _:b439699677 . _:b439699647 . _:b439699676 . . _:b439699679 . _:b8381173 . _:b439699678 . . . _:b439699650 . _:b439699673 . _:b439699666 . _:b439699672 . _:b8381194 "Chk1 is refractory to activation by DNA damage in mitotic cells (Shiromizu et al., >>2006<<); however, when desensitization occurs is unclear." . _:b439699701 . _:b439699675 . _:b439699706 . . _:b439699674 . _:b8381219 . _:b439699688 . _:b439699685 . . _:b439699684 . _:b439699639 . _:b439699687 . _:b8381191 . _:b439699686 . _:b439699681 . _:b8381214 . _:b439699680 . _:b439699683 . _:b8381191 "Cells were counterstained with propidium iodide and analyzed for pH3 fluorescence and DNA content by use of a FACScan flow cytometer (BD) as described previously (Zachos et al., >>2003<<). For S-phase determinations, cells were labeled with 25 \u00B5M BrdU and then fixed and stained using anti-BrdU monoclonal antibody as described previously (Robinson et al., 2006)." . _:b439699682 . _:b439699678 . _:b439699693 . _:b439699692 . _:b8381198 . _:b439699654 . _:b439699695 . _:b439699694 . _:b439699720 . . _:b439699689 . _:b439699688 . . . . _:b439699642 . _:b439699691 . . _:b8381172 . _:b439699705 . _:b439699690 . _:b8381173 . . _:b439699649 . _:b439699701 . . . _:b8381174 . _:b8381208 . _:b439699700 . _:b8381175 . _:b439699685 . _:b439699634 . _:b439699703 . _:b8381168 . . _:b439699702 . _:b8381169 . _:b439699697 . _:b8381190 . _:b8381170 . _:b439699640 . _:b439699696 . _:b8381171 . _:b439699699 . _:b8381180 . . _:b439699635 . . "10.1083%2Fjcb.201003004" . _:b439699698 . _:b8381181 . . _:b439699709 . _:b8381182 . _:b8381216 . _:b439699708 . _:b8381183 . _:b439699711 . _:b8381210 "3 B; Bozulic and Hemmings, >>2009<<). Chemical inhibition of Akt also restored irradiation-induced Chk1 S345 phosphorylation in RO-3306\u2013arrested HCT116 cells (Fig." . . _:b8381176 . _:b439699710 . . _:b8381177 . _:b439699705 . _:b8381178 . _:b439699704 . _:b8381179 . . _:b439699707 . _:b439699706 . _:b439699702 . _:b8381221 . _:b439699729 . _:b8381186 "Cells were irradiated using an Alcyon II CGR MeV cobalt source as described previously (Rainey et al., >>2008<<)." . _:b8381183 _:b8381192 . _:b8381222 . . _:b439699692 . _:b8381183 _:b8381184 . . _:b8381201 . _:b8381183 _:b8381185 . _:b8381183 _:b8381186 . _:b8381183 _:b8381187 . _:b8381183 _:b8381188 . _:b8381169 . _:b8381183 _:b8381189 . _:b8381181 . _:b8381183 _:b8381190 . . _:b8381183 _:b8381191 . . _:b8381179 . _:b439699698 . _:b439699704 . _:b439699705 . _:b439699676 . _:b439699706 . _:b439699707 . . _:b439699708 . _:b439699709 . _:b439699710 . _:b439699711 . _:b439699696 . _:b439699697 . _:b439699698 . _:b439699643 . _:b439699699 .