Dhh1 regulates the G1/S-checkpoint following DNA damage or BRCA1 expression in yeast1

Westmoreland, Tammy J.; Olson, John A.; Saito, Wilfred; Huper, Gudrun; Marks, Jeffrey R.; Bennett, Craig B.

doi:10.1016/s0022-4804(03)00155-0

Cited by 28 publications

(45 citation statements)

References 44 publications

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“…Earlier studies have shown that a DHH1 null mutant is viable but displays sensitivities to stress conditions including heat and DNA-damaging agents such as methyl methane sulfonate, hydroxyurea, and UV irradiation (27,28,30). Mutations to the DEAD-box or residues implicated in RNA binding could not complement the temperature sensitivity of a dhh1⌬ strain (44).…”

Section: Dmentioning

confidence: 99%

“…Dhh1, along with its interacting partners Pat1 and Edc3, is also involved in translational repression of mRNAs and has been shown to sequester RNAs into a non-translating pool that does not undergo degradation (22,25,26). Studies have linked Dhh1 to cell cycle regulation, where it is important for G 1 /S DNA-damage checkpoint recovery in Saccharomyces cerevisiae (27,28). Recent studies have also shown that Dhh1 controls hyphal development (29).…”

mentioning

confidence: 99%

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Intermolecular Interactions within the Abundant DEAD-box Protein Dhh1 Regulate Its Activity in Vivo

Dutta

Zheng

Jain

et al. 2011

Journal of Biological Chemistry

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Dhh1 is a highly conserved DEAD-box protein that has been implicated in many processes involved in mRNA regulation. At least some functions of Dhh1 may be carried out in cytoplasmic foci called processing bodies (P-bodies). Dhh1 was identified initially as a putative RNA helicase based solely on the presence of conserved helicase motifs found in the superfamily 2 (Sf2) of DEXD/H-box proteins. Although initial mutagenesis studies revealed that the signature DEAD-box motif is required for Dhh1 function in vivo, enzymatic (ATPase or helicase) or ATP binding activities of Dhh1 or those of any its many higher eukaryotic orthologues have not been described. Here we provide the first characterization of the biochemical activities of Dhh1. Dhh1 has weaker RNA-dependent ATPase activity than other well characterized DEAD-box helicases. We provide evidence that intermolecular interactions between the N-and C-terminal RecA-like helicase domains restrict its ATPase activity; mutation of residues mediating these interactions enhanced ATP hydrolysis. Interestingly, the interdomain interaction mutant displayed enhanced mRNA turnover, RNA binding, and recruitment into cytoplasmic foci in vivo compared with wild type Dhh1. Also, we demonstrate that the ATPase activity of Dhh1 is not required for it to be recruited into cytoplasmic foci, but it regulates its association with RNA in vivo. We hypothesize that the activity of Dhh1 is restricted by interdomain interactions, which can be regulated by cellular factors to impart stringent control over this very abundant RNA helicase.The regulation of gene expression in eukaryotes is a complex series of events that is well coordinated by different cellular machineries. Central to this process is the regulation of mRNA metabolism. It begins with transcription followed by pre-mRNA processing in the nucleus, nucleo-cytoplasmic transport, translation, and finally, the controlled degradation of mRNA.mRNA degradation is one of the important means of mRNA quality control. In eukaryotic cells two major pathways have evolved for mRNA degradation; they are non-sense-mediated decay, which is deadenylation-independent and the more ubiquitously used deadenylation-dependent pathway (1). Nonsense-mediated decay is the process of choice for quick and efficient removal of transcripts with non-sense codons, unspliced introns, or extended 3Ј-UTR (2). The deadenylationdependent pathway begins with the removal of the 3Ј-poly(A) tail by 3Ј-5Ј exonucleases Ccr4 and Pop2 or poly(A) nuclease (3-7). This is followed by removal of the 5Ј-m 7 -guanosine triphosphate cap by the Dcp2/Dcp1 decapping complex (8, 9). The binding of Dcp1/Dcp2 to the 5Ј cap and decapping are enhanced by a complex of seven Lsm (like-Sm) proteins (Lsm1 through Lsm7), Pat1, Edc3,. The final step in the pathway involves degradation of the decapped and deadenylated RNA by the 5Ј-3Ј exonuclease Xrn1 and by the exosome that acts in the 3Ј-5Ј direction (16,17). Proteins involved in mRNA turnover are localized to distinct foci in the cytoplasm call...

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Section: Dmentioning

confidence: 99%

mentioning

confidence: 99%

Intermolecular Interactions within the Abundant DEAD-box Protein Dhh1 Regulate Its Activity in Vivo

Dutta

Zheng

Jain

et al. 2011

Journal of Biological Chemistry

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“…Brcal induces DNA damage in yeast as measured using a DIN::LacZ reporter of RNR3 transcriptional activation. Deletion of CCR4 dramatically decreases this DNA damage response and implies that less DNA damage is induced by Brcal in ccr4A cells, or alternatively the damage can be repaired more efficiently as the cells continue to progress into later stages of the cell cycle (17). Therefore, we appear to have defined a new GC and/or S phase specific function for Brcal in yeast.…”

Section: Introductionmentioning

confidence: 88%

“…Disruption of G 1 checkpoint functions by deletion of CCR4 or DHH1 allows enhanced survival among cells over expressing Brcal. Enhanced survival is not due to down regulation of Brcal protein expression in ccr4A or dhhlA cells (17). Brcal induces DNA damage in yeast as measured using a DIN::LacZ reporter of RNR3 transcriptional activation.…”

Section: Introductionmentioning

confidence: 99%

“…We have determined that overexpression of Brcal in diploid WT yeast leads to a prolonged arrest in GI and lethality. Furthermore, Brcal expression also activates the DIN: :LacZ reporter construct that indicates the presence of DNA damage or replicative stress (17). We therefore used a yeast functional genomics approach to identify from a collection of IR-sensitive yeast gene deletions (2) (16) or from a pool of genetically tagged deletion strains, those that rescue Brcal -induced lethality.…”

Section: Introductionmentioning

confidence: 99%

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A Functional Genomics Approach to Identify Novel Breast Cancer Gene Targets in Yeast

Bennett¹

2005

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Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302, and ABSTRACT (Maximum 200 Words)We are using the yeast Saccharomyces cerevisiae to identify new cancer gene targets that interact with the tumor suppressor Brcal. Expression of Brcal in diploid WT yeast leads to prolonged Gl arrest and lethality.We identified from a collection of ionizing radiation (IR)-sensitive yeast deletion strains or from a pool of 4746 genetically tagged deletion strains, 34 that rescue Brcal-induced lethality.Two IR resistance genes that rescue Brcal-induced lethality are the transcription factors CCR4 and DHH1. These are checkpoint genes required for cell cycle progression in Gl and S phases following DNA damage.Consistent with a role in radiation resistance, Dhhlp and its highly conserved human ortholog DDX6 were found to physically interact with Brcal in yeast and human cells. Another transcription factor (YAF9) was IR sensitivre and rescued Brcal-induced lethality when deleted.This deletion strain and 19 others were subsequently isolated from the deletion strain pool.Most of these deletions (75%) were IR sensitive and hypersensitive to the toxin zymocin which appears to induce DSB damage by inhibiting transcription. Furthermore, most (85%) of these genes are highly conserved suggesting that the human orthologs may interact with Brcal to maintain genomic stability and suppress the onset of breast cancer. SUBJECT TERMS INTRODUCTION:Cancer is thought to be a multistep process that encompasses many separate molecular mechanisms. Common cancers such as those of the breast and colon have been associated with defects in genes that regulate DNA repair and/or checkpoint functions thereby implicating a loss of genetic stability as a causative factor in these cancers.. In hereditary breast cancer, changes in DNA repair ability have been identified that result from alterations in the gene products Brcal and Brca2. These genes have been found to be pleiotropic tumor suppressors that impact on recombinational repair of DSB damage, cell cycle checkpoint arrest following DNA damage, transcriptional regulatory mechanisms, centrosome duplication and transcription coupled repair of DNA damage (9) (12) (19) (18) (1). We are using the yeast Saccharomyces cerevisiae as a model organism for the identification of the genetic controls associated with the DNA repair and checkpoint functions of Brcal. We have determined that overexpression of Brcal in diploid WT yeast leads to a prolonged arrest in GI and lethality. Furthermo...

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A yeast recombination assay to characterize humanBRCA1missense variants of unknown pathological significance

et al. 2009

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The BRCA1 tumor suppressor gene is found mutated in familial breast cancer. Although many of the mutations are clearly pathological because they give rise to truncated proteins, several missense variants of uncertain pathological consequences have been identified. A novel functional assay to screen for BRCA1 missense variants in a simple genetic system could be very useful for the identification of potentially deleterious mutations. By using two prediction computer programs, Sorting Intolerant from Tolerant (SIFT) and Polymorphism Phenotyping (PolyPhen), seven nonsynonymous missense BRCA1 variants likely disrupting the gene function were selected as potentially deleterious. The budding yeast Saccharomyces cerevisiae (S. cerevisiae) was used to test these cancer-related missense mutations for their ability to affect cell growth and homologous recombination (HR) at the HIS3 and ADE2 loci. The variants localized in the BRCA1 C-Terminus (BRCT) domain did not show any growth inhibition when overexpressed in agreement with previous results. Overexpression of either wild-type BRCA1 or two neutral missense variants did not increase yeast HR but when cancer-related variants were overexpressed a significant increase in recombination was observed. Results clearly showed that this genetic system can be useful to discriminate between neutral and deleterious BRCA1 missense variants.

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Dhh1 regulates the G1/S-checkpoint following DNA damage or BRCA1 expression in yeast1

Cited by 28 publications

References 44 publications

Intermolecular Interactions within the Abundant DEAD-box Protein Dhh1 Regulate Its Activity in Vivo

Intermolecular Interactions within the Abundant DEAD-box Protein Dhh1 Regulate Its Activity in Vivo

A Functional Genomics Approach to Identify Novel Breast Cancer Gene Targets in Yeast

A yeast recombination assay to characterize humanBRCA1missense variants of unknown pathological significance

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