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author:("Surana, uttar")
1.  Staging a recovery from mitotic arrest 
Bioarchitecture  2012;2(2):33-37.
Checkpoint controls, the surveillance pathways that impose “an order of execution” on the major cell cycle events, are critical to the maintenance of genome stability. When cells fail to execute a cellular event or do so erroneously due to misregulation or exposure to genotoxic stresses, these evolutionarily conserved regulatory circuits prevent passage to the subsequent event, thus bringing the cell cycle to a halt. Once the checkpoint stimulus is removed, cells recover from the arrest and eventually resume cell cycle progression. While the activation, execution and maintenance, the three major aspects of the checkpoint controls, have been investigated in detail, the recovery process remains underexplored. It is not clear if cells recover passively upon dissipation of the checkpoint signals or require an active participation by specific effectors. A recent study in the yeast Saccharomyces cerevisiae uncovered two previously unsuspected functions of Cdk1 in efficient recovery from the spindle assembly checkpoint (SAC) imposed arrest. An inability to fulfil these requirements in the absence of Cdk1 makes it virtually impossible for cells to recover from the mitotic arrest. Given the conserved nature of the SAC, these findings may have implications for vertebrate cells.
PMCID: PMC3383719  PMID: 22754627
Cdk1; cell cycle; cell division; checkpoint; mitosis; recovery; spindle; yeast
2.  p38 Mitogen-Activated Protein Kinase Promotes Cell Survival in Response to DNA Damage but Is Not Required for the G2 DNA Damage Checkpoint in Human Cancer Cells▿ †  
Molecular and Cellular Biology  2010;30(15):3816-3826.
p38 mitogen-activated protein kinase (MAPK) is rapidly activated by stresses and is believed to play an important role in the stress response. While Chk1 is known to mediate G2 DNA damage checkpoint control, p38 was also reported to have an essential function in this checkpoint control. Here, we have investigated further the roles of p38 and Chk1 in the G2 DNA damage checkpoint in cancer cells. We find that although p38 activation is strongly induced by DNA damage, its activity is not required for the G2 DNA damage checkpoint. In contrast, Chk1 kinase is responsible for the execution of G2 DNA damage checkpoint control in p53-deficient cells. The inhibition of p38 activity has no effect on Chk1 activation and γ-H2AX expression. Global gene expression profiling of cancer cells in response to tumor necrosis factor alpha (TNF-α) revealed that p38 plays a strong prosurvival role through the coordinated downregulation of proapoptotic genes and upregulation of prosurvival genes. We show that the inhibition of p38 activity during G2 DNA damage checkpoint arrest triggers apoptosis in a p53-independent manner with a concurrent decrease in the level of Bcl2 family proteins. Our results suggest that although p38 MAPK is not required for the G2 DNA damage checkpoint function, it plays an important prosurvival role during the G2 DNA damage checkpoint response through the upregulation of the Bcl2 family proteins.
doi:10.1128/MCB.00949-09
PMCID: PMC2916406  PMID: 20516219
3.  DNA stretching in the nucleosome facilitates alkylation by an intercalating antitumour agent 
Nucleic Acids Research  2009;38(6):2081-2088.
DNA stretching in the nucleosome core can cause dramatic structural distortions, which may influence compaction and factor recognition in chromatin. We find that the base pair unstacking arising from stretching-induced extreme minor groove kinking near the nucleosome centre creates a hot spot for intercalation and alkylation by a novel anticancer compound. This may have far reaching implications for how chromatin structure can influence binding of intercalator species and indicates potential for the development of site selective DNA-binding agents that target unique conformational features of the nucleosome.
doi:10.1093/nar/gkp1174
PMCID: PMC2847236  PMID: 20026584
4.  Inactivation of Cdh1 by synergistic action of Cdk1 and polo kinase is necessary for proper assembly of the mitotic spindle 
Nature cell biology  2008;10(6):665-675.
Separation of duplicated centrosomes (spindle-pole bodies or SPBs in yeast) is a crucial step in the biogenesis of the mitotic spindle. In vertebrates, centrosome separation requires the BimC family kinesin Eg5 and the activities of Cdk1 and polo kinase; however, the roles of these kinases are not fully understood. In Saccharomyces cerevisiae, SPB separation also requires activated Cdk1 and the plus-end kinesins Cin8 (homologous to vertebrate Eg5) and Kip1. Here we report that polo kinase has a role in the separation of SPBs. We show that adequate accumulation of Cin8 and Kip1 requires inactivation of the anaphase-promoting complex-activator Cdh1 through sequential phosphorylation by Cdk1 and polo kinase. In this process, Cdk1 functions as a priming kinase in that Cdk1-mediated phosphorylation creates a binding site for polo kinase, which further phosphorylates Cdh1. Thus, Cdh1 inactivation through the synergistic action of Cdk1 and polo kinase provides a new model for inactivation of cell-cycle effectors.
doi:10.1038/ncb1729
PMCID: PMC2677644  PMID: 18500339
5.  Oscillations of the p53-Akt Network: Implications on Cell Survival and Death 
PLoS ONE  2009;4(2):e4407.
Intracellular protein levels of p53 and MDM2 have been shown to oscillate in response to ionizing radiation (IR), but the physiological significance of these oscillations remains unclear. The p53-MDM2 negative feedback loop – the putative cause of the oscillations – is embedded in a network involving a mutual antagonism (or positive feedback loop) between p53 and AKT. We have shown earlier that this p53-AKT network predicts an all-or-none switching behavior between a pro-survival cellular state (low p53 and high AKT levels) and a pro-apoptotic state (high p53 and low AKT levels). Here, we show that upon exposure to IR, the p53-AKT network can also reproduce the experimentally observed p53 and MDM2 oscillations. The present work is based on the hypothesis that the physiological significance of the experimentally observed oscillations could be found in their role in regulating the switching behavior of the p53-AKT network between pro-survival and pro-apoptotic states. It is shown here that these oscillations are associated with a significant decrease in the threshold level of IR at which switching from a pro-survival to a pro-apoptotic state occurs. Moreover, oscillations in p53 protein levels induce higher levels of expression of p53-target genes compared to non-oscillatory p53, and thus influence cell-fate decisions between cell cycle arrest/DNA damage repair versus apoptosis.
doi:10.1371/journal.pone.0004407
PMCID: PMC2634840  PMID: 19197384
6.  Synthesizing non-natural parts from natural genomic template 
Background
The current knowledge of genes and proteins comes from 'naturally designed' coding and non-coding regions. It would be interesting to move beyond natural boundaries and make user-defined parts. To explore this possibility we made six non-natural proteins in E. coli. We also studied their potential tertiary structure and phenotypic outcomes.
Results
The chosen intergenic sequences were amplified and expressed using pBAD 202/D-TOPO vector. All six proteins showed significantly low similarity to the known proteins in the NCBI protein database. The protein expression was confirmed through Western blot. The endogenous expression of one of the proteins resulted in the cell growth inhibition. The growth inhibition was completely rescued by culturing cells in the inducer-free medium. Computational structure prediction suggests globular tertiary structure for two of the six non-natural proteins synthesized.
Conclusion
To our best knowledge, this is the first study that demonstrates artificial synthesis of non-natural proteins from existing genomic template, their potential tertiary structure and phenotypic outcome. The work presented in this paper opens up a new avenue of investigating fundamental biology. Our approach can also be used to synthesize large numbers of non-natural RNA and protein parts for useful applications.
doi:10.1186/1754-1611-3-2
PMCID: PMC2642765  PMID: 19187561
7.  Disjunction of conjoined twins: Cdk1, Cdh1 and separation of centrosomes 
Cell Division  2006;1:12.
Accurate transmission of chromosomes from parent to progeny cell requires assembly of a bipolar spindle. Centrosomes (spindle pole body in yeast) are critical for the biogenesis of this complex mitotic apparatus since they confer bipolarity on the spindle and serve as the site of microtubule polymerization. In each division cycle, the centrosome is duplicated and the sister-centrosomes move away from each other, forming the two poles of the spindle. While the structure and the duplication of centrosomes have been investigated extensively, the understanding of the control of their segregation remains scant. Recent findings are beginning to yield insights into the regulation of centrosome segregation in yeast and its link to the mitotic kinase.
doi:10.1186/1747-1028-1-12
PMCID: PMC1550386  PMID: 16792804
8.  Inactivation of Mitotic Kinase Triggers Translocation of MEN Components to Mother-Daughter Neck in Yeast 
Molecular Biology of the Cell  2003;14(11):4734-4743.
Chromosome segregation, mitotic exit, and cytokinesis are executed in this order during mitosis. Although a scheme coordinating sister chromatid separation and initiation of mitotic exit has been proposed, the mechanism that temporally links the onset of cytokinesis to mitotic exit is not known. Exit from mitosis is regulated by the mitotic exit network (MEN), which includes a GTPase (Tem1) and various kinases (Cdc15, Cdc5, Dbf2, and Dbf20). Here, we show that Dbf2 and Dbf20 functions are necessary for the execution of cytokinesis. Relocalization of these proteins from spindle pole bodies to mother daughter neck seems to be necessary for this role because cdc15-2 mutant cells, though capable of exiting mitosis at semipermissive temperature, are unable to localize Dbf2 (and Dbf20) to the “neck” and fail to undergo cytokinesis. These cells can assemble and constrict the actomyosin ring normally but are incapable of forming a septum, suggesting that MEN components are critical for the initiation of septum formation. Interestingly, the spindle pole body to neck translocation of Dbf2 and Dbf20 is triggered by the inactivation of mitotic kinase. The requirement of kinase inactivation for translocation of MEN components to the division site thus provides a mechanism that renders mitotic exit a prerequisite for cytokinesis.
doi:10.1091/mbc.E03-04-0238
PMCID: PMC266787  PMID: 12937277
9.  Dependence of pre-mRNA introns on PRP17, a non-essential splicing factor: implications for efficient progression through cell cycle transitions 
Nucleic Acids Research  2003;31(9):2333-2343.
Saccharomyces cerevisiae PRP17 (CDC40) encodes a second-step pre-mRNA splicing factor with a role in cell division. The functions of Prp17 in specific cell cycle transitions were examined using temperature-sensitive alleles in arrest/release experiments. We find that G1/S and G2/M transitions depend on Prp17. G1-synchronized prp17::LEU2 cells arrest at non-permissive temperatures as unbudded haploid cells with low levels of CLN1, CLB5 and RNR1 transcripts. This indicates a Prp17 execution point at or prior to Start. Reduced levels of α-tubulin protein, a mitotic spindle component, underlie the benomyl sensitivity of prp17 mutants and possibly their G2/M arrest. Splicing of TUB1 and TUB3 transcripts, which encode α-tubulin, was analyzed in prp17 and other second-step factor mutants. TUB1 splicing is inefficient in prp17, prp16 and prp22, and marginally affected in prp18, slu7-1 and psf1-1. TUB3 splicing is similarly affected. In vitro splicing with TUB3 pre-mRNA demonstrates a compromised second step in prp17::LEU2 extracts, implicating a direct role for Prp17 in its efficient splicing. Genomic replacement of an intronless TUB1 gene relieves the benomyl sensitivity of prp17 mutants; however, they remain temperature sensitive, implying multiple limiting factors for mitosis. The data suggest that integration of splicing with the cell cycle is important for G1/S and G2/M transitions.
PMCID: PMC154219  PMID: 12711678
10.  Early Expressed Clb Proteins Allow Accumulation of Mitotic Cyclin by Inactivating Proteolytic Machinery during S Phase 
Molecular and Cellular Biology  2001;21(15):5071-5081.
Periodic accumulation and destruction of mitotic cyclins are important for the initiation and termination of M phase. It is known that both APCCdc20 and APCHct1 collaborate to destroy mitotic cyclins during M phase. Here we show that this relationship between anaphase-promoting complex (APC) and Clb proteins is reversed in S phase such that the early Clb kinases (Clb3, Clb4, and Clb5 kinases) inactivate APCHct1 to allow Clb2 accumulation. This alternating antagonism between APC and Clb proteins during S and M phases constitutes an oscillatory system that generates undulations in the levels of mitotic cyclins.
doi:10.1128/MCB.21.15.5071-5081.2001
PMCID: PMC87233  PMID: 11438663
11.  Cdc4, a Protein Required for the Onset of S Phase, Serves an Essential Function during G2/M Transition in Saccharomyces cerevisiae 
Molecular and Cellular Biology  1999;19(8):5512-5522.
Saccharomyces cerevisiae proteins Cdc4 and Cdc20 contain WD40 repeats and participate in proteolytic processes. However, they are thought to act at two different stages of the cell cycle: Cdc4 is involved in the proteolysis of the Cdk inhibitor, Sic1, necessary for G1/S transition, while Cdc20 mediates anaphase-promoting complex-dependent degradation of anaphase inhibitor Pds1, a process necessary for the onset of chromosome segregation. We have isolated three mutant alleles of CDC4 (cdc4-10, cdc4-11, and cdc4-16) which suppress the nuclear division defect of cdc20-1 cells. However, the previously characterized mutation cdc4-1 and a new allele, cdc4-12, do not alleviate the defect of cdc20-1 cells. This genetic interaction suggests an additional role for Cdc4 in G2/M. Reexamination of the cdc4-1 mutant revealed that, in addition to being defective in the onset of S phase, it is also defective in G2/M transition when released from hydroxyurea-induced S-phase arrest. A second function for CDC4 in late S or G2 phase was further confirmed by the observation that cells lacking the CDC4 gene are arrested both at G1/S and at G2/M. We subsequently isolated additional temperature-sensitive mutations in the CDC4 gene (such as cdc4-12) that render the mutant defective in both G1/S and G2/M transitions at the restrictive temperature. While the G1/S block in both cdc4-12 and cdc4Δ mutants is abolished by the deletion of the SIC1 gene (causing the mutants to be arrested predominantly in G2/M), the preanaphase arrest in the cdc4-12 mutant is relieved by the deletion of PDS1. Collectively, these observations suggest that, in addition to its involvement in the initiation of S phase, Cdc4 may also be required for the onset of anaphase.
PMCID: PMC84393  PMID: 10409741
12.  NDD1, a High-Dosage Suppressor of cdc28-1N, Is Essential for Expression of a Subset of Late-S-Phase-Specific Genes in Saccharomyces cerevisiae 
Molecular and Cellular Biology  1999;19(5):3312-3327.
cdc28-1N mutants progress through the G1 and S phases normally at the restrictive temperature but fail to undergo nuclear division. We have isolated a gene, NDD1, which at a high dosage suppresses the nuclear-division defect of cdc28-1N. NDD1 (nuclear division defective) is an essential gene. Its expression during the cell cycle is tightly regulated such that NDD1 RNA is most abundant during the S phase. Cells lacking the NDD1 gene arrest with an elongated bud, a short mitotic spindle, 2N DNA content, and an undivided nucleus, suggesting that its function is required for some aspect of nuclear division. We show that overexpression of Ndd1 results in the upregulation of both CLB1 and CLB2 transcription, suggesting that the suppression of cdc28-1N by NDD1 may be due to an accumulation of these cyclins. Overproduction of Ndd1 also enhances the expression of SWI5, whose transcription, like that of CLB1 and CLB2, is activated in the late S phase. Ndd1 is essential for the expression of CLB1, CLB2, and SWI5, since none of these genes are transcribed in its absence. Both CLB2 expression and its upregulation by NDD1 are mediated by a 240-bp promoter sequence that contains four MCM1-binding sites. However, Ndd1 does not appear to be a component of any of the protein complexes assembled on this DNA fragment, as indicated by gel mobility shift assays. Instead, overexpression of NDD1 prevents the formation of one of the complexes whose appearance correlates with the termination of CLB2 expression in G1. The inability of GAL1 promoter-driven CLB2 to suppress the lethality of NDD1 null mutant suggests that, in addition to CLB1 and CLB2, NDD1 may also be required for the transcription of other genes whose functions are necessary for G2/M transition.
PMCID: PMC84125  PMID: 10207056
13.  Homeostatic control of polo-like kinase-1 engenders non-genetic heterogeneity in G2 checkpoint fidelity and timing 
Nature Communications  2014;5:4048.
The G2 checkpoint monitors DNA damage, preventing mitotic entry until the damage can be resolved. The mechanisms controlling checkpoint recovery are unclear. Here, we identify non-genetic heterogeneity in the fidelity and timing of damage-induced G2 checkpoint enforcement in individual cells from the same population. Single-cell fluorescence imaging reveals that individual damaged cells experience varying durations of G2 arrest, and recover with varying levels of remaining checkpoint signal or DNA damage. A gating mechanism dependent on polo-like kinase-1 (PLK1) activity underlies this heterogeneity. PLK1 activity continually accumulates from initial levels in G2-arrested cells, at a rate inversely correlated to checkpoint activation, until it reaches a threshold allowing mitotic entry regardless of remaining checkpoint signal or DNA damage. Thus, homeostatic control of PLK1 by the dynamic opposition between checkpoint signalling and pro-mitotic activities heterogeneously enforces the G2 checkpoint in each individual cell, with implications for cancer pathogenesis and therapy.
Cells exposed to DNA damage delay mitotic entry to allow repair. Liang et al. unexpectedly find that the duration of arrest and the completeness of repair vary from cell to cell, determined by progressively increasing polo-like kinase-1 activity, which must pass a threshold to trigger mitosis.
doi:10.1038/ncomms5048
PMCID: PMC4059941  PMID: 24893992

Results 1-13 (13)