Saravana Selvanathan - Academia.edu (original) (raw)

Papers by Saravana Selvanathan

YK-4-279 and TK-216 combinations. For each combination, the table shows the median Combination In... more YK-4-279 and TK-216 combinations. For each combination, the table shows the median Combination Index (CI) obtained after exposing cell lines to increasing concentration of two compounds at 8 x 8 concentrations of each compound (removing concentrations that give 10% or less of proliferation already with the single agent), as previously performed (3).

Gene expression data after YK-4-279 or TK-216 treatment (18h) in U2932, TMD8, OCILY10 and SUDHL2.... more Gene expression data after YK-4-279 or TK-216 treatment (18h) in U2932, TMD8, OCILY10 and SUDHL2. A) Supervised analysis of transcriptome after treatment. B) Gene-sets significantly enriched after treatment.

Gene expression data after YK-4-279 treatment (4-8h) in U2932 and TMD8. A) Supervised analysis of... more Gene expression data after YK-4-279 treatment (4-8h) in U2932 and TMD8. A) Supervised analysis of gene expression profiling after treatment; B) Gene-sets significantly enriched after treatment; C) SPIB/IRF4/lenalidomide regulated gene-sets significantly enriched after treatment.

Gene expression data after TK-216 treatment (8h) in U2932. A) Supervised analysis of transcriptom... more Gene expression data after TK-216 treatment (8h) in U2932. A) Supervised analysis of transcriptome after treatment B) Gene-sets significantly enriched after treatment. C) IRF4/SPIB and lenalidomide related gene-sets significantly enriched after treatment.

Purpose:Transcription factors are commonly deregulated in cancer, and they have been widely consi... more Purpose:Transcription factors are commonly deregulated in cancer, and they have been widely considered as difficult to target due to their nonenzymatic mechanism of action. Altered expression levels of members of the ETS-transcription factors are often observed in many different tumors, including lymphomas. Here, we characterized two small molecules, YK-4-279 and its clinical derivative, TK-216, targeting ETS factors via blocking the protein–protein interaction with RNA helicases, for their antilymphoma activity.Experimental Design:The study included preclinical in vitro activity screening on a large panel of cell lines, both as single agent and in combination; validation experiments on in vivo models; and transcriptome and coimmunoprecipitation experiments.Results:YK-4-279 and TK-216 demonstrated an antitumor activity across several lymphoma cell lines, which we validated in vivo. We observed synergistic activity when YK-4-279 and TK-216 were combined with the BCL2 inhibitor veneto...

Science Advances

Cancers are often defined by the dysregulation of specific transcriptional programs; however, the... more Cancers are often defined by the dysregulation of specific transcriptional programs; however, the importance of global transcriptional changes is less understood. Hypertranscription is the genome-wide increase in RNA output. Hypertranscription’s prevalence, underlying drivers, and prognostic significance are undefined in primary human cancer. This is due, in part, to limitations of expression profiling methods, which assume equal RNA output between samples. Here, we developed a computational method to directly measure hypertranscription in 7494 human tumors, spanning 31 cancer types. Hypertranscription is ubiquitous across cancer, especially in aggressive disease. It defines patient subgroups with worse survival, even within well-established subtypes. Our data suggest that loss of transcriptional suppression underpins the hypertranscriptional phenotype. Single-cell analysis reveals hypertranscriptional clones, which dominate transcript production regardless of their size. Last, pati...

Cancer Research

Cancers are often defined by the dysregulation of specific transcriptional programs; however, the... more Cancers are often defined by the dysregulation of specific transcriptional programs; however, the importance of global transcriptional changes is less understood. Hypertranscription is the genome-wide increase in RNA output. Hypertranscription’s prevalence, underlying drivers and prognostic significance are undefined in primary human cancer. This is due in part to limitations of expression profiling methods, which assume equal RNA output between samples. Here, we developed a computational method to directly measure hypertranscription in 7,494 human tumors, spanning 31 cancer types. Hypertranscription is ubiquitous across cancer, especially in aggressive disease. It defines patient subgroups with worse survival, even within well-established subtypes. Our data suggest that loss of transcriptional suppression underpins the hypertranscriptional phenotype. Single-cell analysis reveals hypertranscriptional clones, which dominate transcript production regardless of their size. Finally, pat...

NAR Cancer, 2022

Ewing sarcoma (EwS) is a small round blue cell tumor and is the second most frequent pediatric bo... more Ewing sarcoma (EwS) is a small round blue cell tumor and is the second most frequent pediatric bone cancer. 85% of EwS tumors express the fusion oncoprotein EWS-FLI1, the product of a t(11;22) reciprocal translocation. Prior work has indicated that transcription regulation alone does not fully describe the oncogenic capacity of EWS-FLI1, nor does it provide an effective means to stratify patient tumors. Research using EwS cell lines and patient samples has suggested that EWS-FLI1 also disrupts mRNA biogenesis. In this work we both describe the underlying characteristics of mRNA that are aberrantly spliced in EwS tumor samples as well as catalogue mRNA splicing events across other pediatric tumor types. Here, we also use short- and long-read sequencing to identify cis-factors that contribute to splicing profiles we observe in Ewing sarcoma. Our analysis suggests that GC content upstream of cassette exons is a defining factor of mRNA splicing in EwS. We also describe specific splicing...

Molecular Medicine Reports

Nucleic Acids Research

Connections between epigenetic reprogramming and transcription or splicing create novel mechanist... more Connections between epigenetic reprogramming and transcription or splicing create novel mechanistic networks that can be targeted with tailored therapies. Multiple subunits of the chromatin remodeling BAF complex, including ARID1A, play a role in oncogenesis, either as tumor suppressors or oncogenes. Recent work demonstrated that EWS–FLI1, the oncogenic driver of Ewing sarcoma (ES), plays a role in chromatin regulation through interactions with the BAF complex. However, the specific BAF subunits that interact with EWS–FLI1 and the precise role of the BAF complex in ES oncogenesis remain unknown. In addition to regulating transcription, EWS–FLI1 also alters the splicing of many mRNA isoforms, but the role of splicing modulation in ES oncogenesis is not well understood. We have identified a direct connection between the EWS–FLI1 protein and ARID1A isoform protein variant ARID1A-L. We demonstrate here that ARID1A-L is critical for ES maintenance and supports oncogenic transformation. W...

Science signaling, Jan 3, 2017

Ewing's sarcoma (ES) is a rare and highly malignant cancer that grows in the bones or surroun... more Ewing's sarcoma (ES) is a rare and highly malignant cancer that grows in the bones or surrounding tissues mostly affecting adolescents and young adults. A chimeric fusion between the RNA binding protein EWS and the ETS family transcription factor FLI1 (EWS-FLI1), which is generated from a chromosomal translocation, is implicated in driving most ES cases by modulation of transcription and alternative splicing. The small-molecule YK-4-279 inhibits EWS-FLI1 function and induces apoptosis in ES cells. We aimed to identify both the underlying mechanism of the drug and potential combination therapies that might enhance its antitumor activity. We tested 69 anticancer drugs in combination with YK-4-279 and found that vinca alkaloids exhibited synergy with YK-4-279 in five ES cell lines. The combination of YK-4-279 and vincristine reduced tumor burden and increased survival in mice bearing ES xenografts. We determined that independent drug-induced events converged to cause this synergist...

Molecular and Cellular Biology, 2015

Ezrin is a key regulator of cancer metastasis that links extracellular matrix to the actin cytosk... more Ezrin is a key regulator of cancer metastasis that links extracellular matrix to the actin cytoskeleton and regulates cell morphology and motility. We discovered a small molecule inhibitor, NSC305787 that directly binds to ezrin and inhibits its function. In this study, we used a nano LC-MS/MS-based proteomic approach to identify ezrin-interacting proteins that are competed away by NSC305787. A large number of the proteins that interact with ezrin were implicated in protein translation and stress granule dynamics. We validated direct interaction between ezrin and the RNA helicase DDX3, and NSC305787 blocked this interaction. Down-regulation or long-term pharmacological inhibition of ezrin led to reduced DDX3 protein levels without changes inDDX3mRNA. Ectopic overexpression of ezrin in low ezrin-expressing osteosarcoma cells caused a notable increase in DDX3 protein levels. Ezrin inhibited RNA helicase activity of DDX3 but increased its ATPase activity. Our data suggest that ezrin co...

Proceedings of the National Academy of Sciences, 2015

The synthesis and processing of mRNA, from transcription to translation initiation, often require... more The synthesis and processing of mRNA, from transcription to translation initiation, often requires splicing of intragenic material. The final mRNA composition varies based on proteins that modulate splice site selection. EWS-FLI1 is an Ewing sarcoma (ES) oncoprotein with an interactome that we demonstrate to have multiple partners in spliceosomal complexes. We evaluate the effect of EWS-FLI1 on posttranscriptional gene regulation using both exon array and RNA-seq. Genes that potentially regulate oncogenesis, including CLK1, CASP3, PPFIBP1, and TERT, validate as alternatively spliced by EWS-FLI1. In a CLIP-seq experiment, we find that EWS-FLI1 RNA-binding motifs most frequently occur adjacent to intron–exon boundaries. EWS-FLI1 also alters splicing by directly binding to known splicing factors including DDX5, hnRNP K, and PRPF6. Reduction of EWS-FLI1 produces an isoform of γ-TERT that has increased telomerase activity compared with wild-type (WT) TERT. The small molecule YK-4–279 is ...

Oncotarget, 2012

Oncogenic fusion proteins, such as EWS-FLI1, are excellent therapeutic targets as they are only l... more Oncogenic fusion proteins, such as EWS-FLI1, are excellent therapeutic targets as they are only located within the tumor. However, there are currently no agents targeted toward transcription factors, which are often considered to be 'undruggable.' A considerable body of evidence is accruing that refutes this claim based upon the intrinsic disorder of transcription factors. Our previous studies show that RNA Helicase A (RHA) enhances the oncogenesis of EWS-FLI1, a putative intrinsically disordered protein. Interruption of this protein-protein complex by small molecule inhibitors validates this interaction as a unique therapeutic target. Single enantiomer activity from a chiral compound has been recognized as strong evidence for specificity in a small molecule-protein interaction. Our compound, YK-4-279, has a chiral center and can be separated into two enantiomers by chiral HPLC. We show that there is a significant difference in activity between the two enantiomers. (S)-YK-4-...

Journal of Biological Chemistry, 2010

Schizosaccharomyces pombe Dss1p and its homologs function in multiple cellular processes includin... more Schizosaccharomyces pombe Dss1p and its homologs function in multiple cellular processes including recombinational repair of DNA and nuclear export of messenger RNA. We found that Tap-tagged Rad24p, a member of the 14-3-3 class of proteins, co-purified Dss1p along with mitotic activator Cdc25p, messenger RNA export/cell cycle factor Rae1p, 19 S proteasomal factors, and recombination protein Rhp51p (a Rad51p homolog). Using chromatin immunoprecipitation, we found that Dss1p recruited Rad24p and Rae1p to the double-strand break (DSB) sites. Furthermore, Cdc25p also recruited to the DSB site, and its recruitment was dependent on Dss1p, Rad24p, and the protein kinase Chk1p. Following DSB, all nuclear Cdc25p was found to be chromatin-associated. We found that Dss1p and Rae1p have a DNA damage checkpoint function, and upon treatment with UV light ⌬dss1 cells entered mitosis prematurely with indistinguishable timing from ⌬rad24 cells. Taken together, these results suggest that Dss1p plays a critical role in linking repair and checkpoint factors to damaged DNA sites by specifically recruiting Rad24p and Cdc25p to the DSBs. We suggest that the sequestration of Cdc25p to DNA damage sites could provide a mechanism for S. pombe cells to arrest at G 2 /M boundary in response to DNA damage. Eukaryotic cells respond to double-strand breaks (DSBs) 3 within DNA by activating DNA damage checkpoint proteins that send signals to the cells, ultimately resulting in cell cycle arrest. The arrest allows DNA damage to be repaired by the proteins of the DNA repair pathway before cells can enter mitosis (1, 2). In Schizosaccharomyces pombe, entry into mitosis at the G 2 /M boundary is regulated by the phosphorylation status of the mitotic regulator Cdc2p at the tyrosine 15 residue (3). The cells are maintained in G 2 by phosphorylation of Cdc2p by Wee1p and Mik1p kinases, whereas their entry into mitosis is triggered by dephosphorylation of Cdc2p by the Cdc25p phosphatase (3). Dss1p, or its Saccharomyces cerevisiae homolog Sem1p, is a small acidic protein that is required for efficient DNA repair and the nuclear export of messenger RNA (mRNA) (4-7). Dss1p is a co-factor for human breast cancer susceptibility protein BRCA2 (8). In Ustilago maydis, a Dss1p homolog is a cofactor for Brh2p, a homolog of the human BRCA2 (9). The association between Dss1p and BRCA2 regulates the function of recombination-repair protein Rad51p (a homolog of the bacterial RecA; Rhp51p in S. pombe) (10, 11). So far the homologs of BRCA2/Brh2p have not been reported in either S. cerevisiae or in S. pombe. S. cerevisiae Sem1p was shown to recruit to DSB sites following a pattern similar to Rad51p, with high enrichment around the break site and gradually decreasing away from the break in both directions (12). Both Dss1p and Sem1p were shown to associate with the 19 S subunit of the 26 S proteasomes (12-14). It was suggested that the function of Sem1p involves regulating the function of the proteasome complex in DNA repair (12). So far the corresponding role of S. pombe Dss1p in DNA recombination-repair has not been studied. S. pombe Rad24p belongs to the 14-3-3 family of proteins that play a significant role as checkpoint factors in monitoring DNA damage in the G 2 phase of the cell cycle (15-18). Their role in the cell cycle was first demonstrated in S. pombe, where the products of the rad24 and rad25 genes were shown to possess a DNA damage checkpoint function (19). Neither the rad24 nor the rad25 gene is essential for growth, but simultaneous loss of both genes is lethal (19). The loss of rad24, but not rad25, leads to premature entry of cells into mitosis, resulting in small, round cells. In addition, the loss of rad24 renders cells highly sensitive to DNA-damaging agents, whereas a rad25 null strain is only modestly sensitive (19). In response to DNA damage in the G 2 stage, activated Chk1p kinase phosphorylates Cdc25p. Recently Mek1p was shown to phosphorylate Cdc25p independent of Chk1p (20). Rad24p binds phosphorylated Cdc25p and apparently blocks a nuclear localization signal within Cdc25p. The Rad24p-Cdc25p complex exits the nucleus by using a dedicated nuclear export pathway. It was originally suggested that the "nuclear exclusion" of Cdc25p prevents the

YK-4-279 and TK-216 combinations. For each combination, the table shows the median Combination In... more YK-4-279 and TK-216 combinations. For each combination, the table shows the median Combination Index (CI) obtained after exposing cell lines to increasing concentration of two compounds at 8 x 8 concentrations of each compound (removing concentrations that give 10% or less of proliferation already with the single agent), as previously performed (3).

Gene expression data after YK-4-279 or TK-216 treatment (18h) in U2932, TMD8, OCILY10 and SUDHL2.... more Gene expression data after YK-4-279 or TK-216 treatment (18h) in U2932, TMD8, OCILY10 and SUDHL2. A) Supervised analysis of transcriptome after treatment. B) Gene-sets significantly enriched after treatment.

Gene expression data after YK-4-279 treatment (4-8h) in U2932 and TMD8. A) Supervised analysis of... more Gene expression data after YK-4-279 treatment (4-8h) in U2932 and TMD8. A) Supervised analysis of gene expression profiling after treatment; B) Gene-sets significantly enriched after treatment; C) SPIB/IRF4/lenalidomide regulated gene-sets significantly enriched after treatment.

Gene expression data after TK-216 treatment (8h) in U2932. A) Supervised analysis of transcriptom... more Gene expression data after TK-216 treatment (8h) in U2932. A) Supervised analysis of transcriptome after treatment B) Gene-sets significantly enriched after treatment. C) IRF4/SPIB and lenalidomide related gene-sets significantly enriched after treatment.

Purpose:Transcription factors are commonly deregulated in cancer, and they have been widely consi... more Purpose:Transcription factors are commonly deregulated in cancer, and they have been widely considered as difficult to target due to their nonenzymatic mechanism of action. Altered expression levels of members of the ETS-transcription factors are often observed in many different tumors, including lymphomas. Here, we characterized two small molecules, YK-4-279 and its clinical derivative, TK-216, targeting ETS factors via blocking the protein–protein interaction with RNA helicases, for their antilymphoma activity.Experimental Design:The study included preclinical in vitro activity screening on a large panel of cell lines, both as single agent and in combination; validation experiments on in vivo models; and transcriptome and coimmunoprecipitation experiments.Results:YK-4-279 and TK-216 demonstrated an antitumor activity across several lymphoma cell lines, which we validated in vivo. We observed synergistic activity when YK-4-279 and TK-216 were combined with the BCL2 inhibitor veneto...

Science Advances

Cancers are often defined by the dysregulation of specific transcriptional programs; however, the... more Cancers are often defined by the dysregulation of specific transcriptional programs; however, the importance of global transcriptional changes is less understood. Hypertranscription is the genome-wide increase in RNA output. Hypertranscription’s prevalence, underlying drivers, and prognostic significance are undefined in primary human cancer. This is due, in part, to limitations of expression profiling methods, which assume equal RNA output between samples. Here, we developed a computational method to directly measure hypertranscription in 7494 human tumors, spanning 31 cancer types. Hypertranscription is ubiquitous across cancer, especially in aggressive disease. It defines patient subgroups with worse survival, even within well-established subtypes. Our data suggest that loss of transcriptional suppression underpins the hypertranscriptional phenotype. Single-cell analysis reveals hypertranscriptional clones, which dominate transcript production regardless of their size. Last, pati...

Cancer Research

Cancers are often defined by the dysregulation of specific transcriptional programs; however, the... more Cancers are often defined by the dysregulation of specific transcriptional programs; however, the importance of global transcriptional changes is less understood. Hypertranscription is the genome-wide increase in RNA output. Hypertranscription’s prevalence, underlying drivers and prognostic significance are undefined in primary human cancer. This is due in part to limitations of expression profiling methods, which assume equal RNA output between samples. Here, we developed a computational method to directly measure hypertranscription in 7,494 human tumors, spanning 31 cancer types. Hypertranscription is ubiquitous across cancer, especially in aggressive disease. It defines patient subgroups with worse survival, even within well-established subtypes. Our data suggest that loss of transcriptional suppression underpins the hypertranscriptional phenotype. Single-cell analysis reveals hypertranscriptional clones, which dominate transcript production regardless of their size. Finally, pat...

NAR Cancer, 2022

Ewing sarcoma (EwS) is a small round blue cell tumor and is the second most frequent pediatric bo... more Ewing sarcoma (EwS) is a small round blue cell tumor and is the second most frequent pediatric bone cancer. 85% of EwS tumors express the fusion oncoprotein EWS-FLI1, the product of a t(11;22) reciprocal translocation. Prior work has indicated that transcription regulation alone does not fully describe the oncogenic capacity of EWS-FLI1, nor does it provide an effective means to stratify patient tumors. Research using EwS cell lines and patient samples has suggested that EWS-FLI1 also disrupts mRNA biogenesis. In this work we both describe the underlying characteristics of mRNA that are aberrantly spliced in EwS tumor samples as well as catalogue mRNA splicing events across other pediatric tumor types. Here, we also use short- and long-read sequencing to identify cis-factors that contribute to splicing profiles we observe in Ewing sarcoma. Our analysis suggests that GC content upstream of cassette exons is a defining factor of mRNA splicing in EwS. We also describe specific splicing...

Molecular Medicine Reports

Nucleic Acids Research

Connections between epigenetic reprogramming and transcription or splicing create novel mechanist... more Connections between epigenetic reprogramming and transcription or splicing create novel mechanistic networks that can be targeted with tailored therapies. Multiple subunits of the chromatin remodeling BAF complex, including ARID1A, play a role in oncogenesis, either as tumor suppressors or oncogenes. Recent work demonstrated that EWS–FLI1, the oncogenic driver of Ewing sarcoma (ES), plays a role in chromatin regulation through interactions with the BAF complex. However, the specific BAF subunits that interact with EWS–FLI1 and the precise role of the BAF complex in ES oncogenesis remain unknown. In addition to regulating transcription, EWS–FLI1 also alters the splicing of many mRNA isoforms, but the role of splicing modulation in ES oncogenesis is not well understood. We have identified a direct connection between the EWS–FLI1 protein and ARID1A isoform protein variant ARID1A-L. We demonstrate here that ARID1A-L is critical for ES maintenance and supports oncogenic transformation. W...

Science signaling, Jan 3, 2017

Ewing's sarcoma (ES) is a rare and highly malignant cancer that grows in the bones or surroun... more Ewing's sarcoma (ES) is a rare and highly malignant cancer that grows in the bones or surrounding tissues mostly affecting adolescents and young adults. A chimeric fusion between the RNA binding protein EWS and the ETS family transcription factor FLI1 (EWS-FLI1), which is generated from a chromosomal translocation, is implicated in driving most ES cases by modulation of transcription and alternative splicing. The small-molecule YK-4-279 inhibits EWS-FLI1 function and induces apoptosis in ES cells. We aimed to identify both the underlying mechanism of the drug and potential combination therapies that might enhance its antitumor activity. We tested 69 anticancer drugs in combination with YK-4-279 and found that vinca alkaloids exhibited synergy with YK-4-279 in five ES cell lines. The combination of YK-4-279 and vincristine reduced tumor burden and increased survival in mice bearing ES xenografts. We determined that independent drug-induced events converged to cause this synergist...

Molecular and Cellular Biology, 2015

Ezrin is a key regulator of cancer metastasis that links extracellular matrix to the actin cytosk... more Ezrin is a key regulator of cancer metastasis that links extracellular matrix to the actin cytoskeleton and regulates cell morphology and motility. We discovered a small molecule inhibitor, NSC305787 that directly binds to ezrin and inhibits its function. In this study, we used a nano LC-MS/MS-based proteomic approach to identify ezrin-interacting proteins that are competed away by NSC305787. A large number of the proteins that interact with ezrin were implicated in protein translation and stress granule dynamics. We validated direct interaction between ezrin and the RNA helicase DDX3, and NSC305787 blocked this interaction. Down-regulation or long-term pharmacological inhibition of ezrin led to reduced DDX3 protein levels without changes inDDX3mRNA. Ectopic overexpression of ezrin in low ezrin-expressing osteosarcoma cells caused a notable increase in DDX3 protein levels. Ezrin inhibited RNA helicase activity of DDX3 but increased its ATPase activity. Our data suggest that ezrin co...

Proceedings of the National Academy of Sciences, 2015

The synthesis and processing of mRNA, from transcription to translation initiation, often require... more The synthesis and processing of mRNA, from transcription to translation initiation, often requires splicing of intragenic material. The final mRNA composition varies based on proteins that modulate splice site selection. EWS-FLI1 is an Ewing sarcoma (ES) oncoprotein with an interactome that we demonstrate to have multiple partners in spliceosomal complexes. We evaluate the effect of EWS-FLI1 on posttranscriptional gene regulation using both exon array and RNA-seq. Genes that potentially regulate oncogenesis, including CLK1, CASP3, PPFIBP1, and TERT, validate as alternatively spliced by EWS-FLI1. In a CLIP-seq experiment, we find that EWS-FLI1 RNA-binding motifs most frequently occur adjacent to intron–exon boundaries. EWS-FLI1 also alters splicing by directly binding to known splicing factors including DDX5, hnRNP K, and PRPF6. Reduction of EWS-FLI1 produces an isoform of γ-TERT that has increased telomerase activity compared with wild-type (WT) TERT. The small molecule YK-4–279 is ...

Oncotarget, 2012

Oncogenic fusion proteins, such as EWS-FLI1, are excellent therapeutic targets as they are only l... more Oncogenic fusion proteins, such as EWS-FLI1, are excellent therapeutic targets as they are only located within the tumor. However, there are currently no agents targeted toward transcription factors, which are often considered to be 'undruggable.' A considerable body of evidence is accruing that refutes this claim based upon the intrinsic disorder of transcription factors. Our previous studies show that RNA Helicase A (RHA) enhances the oncogenesis of EWS-FLI1, a putative intrinsically disordered protein. Interruption of this protein-protein complex by small molecule inhibitors validates this interaction as a unique therapeutic target. Single enantiomer activity from a chiral compound has been recognized as strong evidence for specificity in a small molecule-protein interaction. Our compound, YK-4-279, has a chiral center and can be separated into two enantiomers by chiral HPLC. We show that there is a significant difference in activity between the two enantiomers. (S)-YK-4-...

Journal of Biological Chemistry, 2010

Schizosaccharomyces pombe Dss1p and its homologs function in multiple cellular processes includin... more Schizosaccharomyces pombe Dss1p and its homologs function in multiple cellular processes including recombinational repair of DNA and nuclear export of messenger RNA. We found that Tap-tagged Rad24p, a member of the 14-3-3 class of proteins, co-purified Dss1p along with mitotic activator Cdc25p, messenger RNA export/cell cycle factor Rae1p, 19 S proteasomal factors, and recombination protein Rhp51p (a Rad51p homolog). Using chromatin immunoprecipitation, we found that Dss1p recruited Rad24p and Rae1p to the double-strand break (DSB) sites. Furthermore, Cdc25p also recruited to the DSB site, and its recruitment was dependent on Dss1p, Rad24p, and the protein kinase Chk1p. Following DSB, all nuclear Cdc25p was found to be chromatin-associated. We found that Dss1p and Rae1p have a DNA damage checkpoint function, and upon treatment with UV light ⌬dss1 cells entered mitosis prematurely with indistinguishable timing from ⌬rad24 cells. Taken together, these results suggest that Dss1p plays a critical role in linking repair and checkpoint factors to damaged DNA sites by specifically recruiting Rad24p and Cdc25p to the DSBs. We suggest that the sequestration of Cdc25p to DNA damage sites could provide a mechanism for S. pombe cells to arrest at G 2 /M boundary in response to DNA damage. Eukaryotic cells respond to double-strand breaks (DSBs) 3 within DNA by activating DNA damage checkpoint proteins that send signals to the cells, ultimately resulting in cell cycle arrest. The arrest allows DNA damage to be repaired by the proteins of the DNA repair pathway before cells can enter mitosis (1, 2). In Schizosaccharomyces pombe, entry into mitosis at the G 2 /M boundary is regulated by the phosphorylation status of the mitotic regulator Cdc2p at the tyrosine 15 residue (3). The cells are maintained in G 2 by phosphorylation of Cdc2p by Wee1p and Mik1p kinases, whereas their entry into mitosis is triggered by dephosphorylation of Cdc2p by the Cdc25p phosphatase (3). Dss1p, or its Saccharomyces cerevisiae homolog Sem1p, is a small acidic protein that is required for efficient DNA repair and the nuclear export of messenger RNA (mRNA) (4-7). Dss1p is a co-factor for human breast cancer susceptibility protein BRCA2 (8). In Ustilago maydis, a Dss1p homolog is a cofactor for Brh2p, a homolog of the human BRCA2 (9). The association between Dss1p and BRCA2 regulates the function of recombination-repair protein Rad51p (a homolog of the bacterial RecA; Rhp51p in S. pombe) (10, 11). So far the homologs of BRCA2/Brh2p have not been reported in either S. cerevisiae or in S. pombe. S. cerevisiae Sem1p was shown to recruit to DSB sites following a pattern similar to Rad51p, with high enrichment around the break site and gradually decreasing away from the break in both directions (12). Both Dss1p and Sem1p were shown to associate with the 19 S subunit of the 26 S proteasomes (12-14). It was suggested that the function of Sem1p involves regulating the function of the proteasome complex in DNA repair (12). So far the corresponding role of S. pombe Dss1p in DNA recombination-repair has not been studied. S. pombe Rad24p belongs to the 14-3-3 family of proteins that play a significant role as checkpoint factors in monitoring DNA damage in the G 2 phase of the cell cycle (15-18). Their role in the cell cycle was first demonstrated in S. pombe, where the products of the rad24 and rad25 genes were shown to possess a DNA damage checkpoint function (19). Neither the rad24 nor the rad25 gene is essential for growth, but simultaneous loss of both genes is lethal (19). The loss of rad24, but not rad25, leads to premature entry of cells into mitosis, resulting in small, round cells. In addition, the loss of rad24 renders cells highly sensitive to DNA-damaging agents, whereas a rad25 null strain is only modestly sensitive (19). In response to DNA damage in the G 2 stage, activated Chk1p kinase phosphorylates Cdc25p. Recently Mek1p was shown to phosphorylate Cdc25p independent of Chk1p (20). Rad24p binds phosphorylated Cdc25p and apparently blocks a nuclear localization signal within Cdc25p. The Rad24p-Cdc25p complex exits the nucleus by using a dedicated nuclear export pathway. It was originally suggested that the "nuclear exclusion" of Cdc25p prevents the