ABSTRACT
Pyrimidines are important nucleic acid precursors which are constantly synthesized, degraded, and rebuilt in the cell. Four degradation pathways, two of which are found in eukaryotes, have been described. One of them, the URC pathway, has been initially discovered in our laboratory in the yeast Lachancea kluyveri. Here, we present the global changes in gene expression in L. kluyveri in response to different nitrogen sources, including uracil, uridine, dihydrouracil, and ammonia. The expression pattern of the known URC genes, URC1-6, helped to identify nine putative novel URC genes with a similar expression pattern. The microarray analysis provided evidence that both the URC and PYD genes are under nitrogen catabolite repression in L. kluyveri and are induced by uracil or dihydrouracil, respectively. We determined the function of URC8, which was found to catalyze the reduction of malonate semialdehyde to 3-hydroxypropionate, the final degradation product of the pathway. The other eight genes studied were all putative permeases. Our analysis of double deletion strains showed that the L. kluyveri Fui1p protein transported uridine, just like its homolog in Saccharomyces cerevisiae, but we demonstrated that is was not the only uridine transporter in L. kluyveri. We also showed that the L. kluyveri homologs of DUR3 and FUR4 do not have the same function that they have in S. cerevisiae, where they transport urea and uracil, respectively. In L. kluyveri, both of these deletion strains grew normally on uracil and urea.
Subject(s)
Fungal Proteins/metabolism , Genome, Fungal , Nucleoside Transport Proteins/metabolism , Saccharomyces/metabolism , Uracil/metabolism , Catabolite Repression , Fungal Proteins/genetics , Gene Expression Profiling , Nitrogen/metabolism , Nucleoside Transport Proteins/genetics , Saccharomyces/geneticsABSTRACT
The molecular events occurring following the disruption of DNA replication forks are poorly characterized, despite extensive use of replication inhibitors such as hydroxyurea in the treatment of malignancies. Here, we identify a key role for the FBH1 helicase in mediating DNA double-strand break formation following replication inhibition. We show that FBH1-deficient cells are resistant to killing by hydroxyurea, and exhibit impaired activation of the pro-apoptotic factor p53, consistent with decreased DNA double-strand break formation. Similar findings were obtained in murine ES cells carrying disrupted alleles of Fbh1. We also show that FBH1 through its helicase activity co-operates with the MUS81 nuclease in promoting the endonucleolytic DNA cleavage following prolonged replication stress. Accordingly, MUS81 and EME1-depleted cells show increased resistance to the cytotoxic effects of replication stress. Our data suggest that FBH1 helicase activity is required to eliminate cells with excessive replication stress through the generation of MUS81-induced DNA double-strand breaks.
Subject(s)
DNA Breaks, Double-Stranded , DNA Helicases/metabolism , DNA Replication , DNA-Binding Proteins/metabolism , Endonucleases/metabolism , F-Box Proteins/metabolism , Stress, Physiological , Alleles , Animals , Blotting, Southern , Cell Death/drug effects , Cell Line, Tumor , DNA Breaks, Double-Stranded/drug effects , DNA Replication/drug effects , Doxycycline/pharmacology , Embryonic Stem Cells/cytology , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/enzymology , Humans , Mice , RNA, Small Interfering/metabolism , Signal Transduction/drug effects , Stress, Physiological/drug effectsABSTRACT
Activation of oncogenes or inhibition of WEE1 kinase deregulates cyclin-dependent kinase (CDK) activity and leads to replication stress; however, the underlying mechanism is not understood. We now show that elevation of CDK activity by inhibition of WEE1 kinase rapidly increases initiation of replication. This leads to nucleotide shortage and reduces replication fork speed, which is followed by SLX4/MUS81-mediated DNA double-strand breakage. Fork speed is normalized and DNA double-strand break (DSB) formation is suppressed when CDT1, a key factor for replication initiation, is depleted. Furthermore, addition of nucleosides counteracts the effects of unscheduled CDK activity on fork speed and DNA DSB formation. Finally, we show that WEE1 regulates the ionizing radiation (IR)-induced S-phase checkpoint, consistent with its role in control of replication initiation. In conclusion, these results suggest that deregulated CDK activity, such as that occurring following inhibition of WEE1 kinase or activation of oncogenes, induces replication stress and loss of genomic integrity through increased firing of replication origins and subsequent nucleotide shortage.
Subject(s)
CDC2 Protein Kinase/metabolism , Cell Cycle Proteins/metabolism , DNA Replication , Genome, Human , Genomic Instability , Nuclear Proteins/metabolism , Protein-Tyrosine Kinases/metabolism , CDC2 Protein Kinase/antagonists & inhibitors , Cell Line , DNA Breaks, Double-Stranded , Humans , Nucleotides/metabolism , Recombinases/metabolism , S Phase Cell Cycle Checkpoints/radiation effectsABSTRACT
Lens epithelium-derived growth factor p75 splice variant (LEDGF) is a chromatin-binding protein known for its antiapoptotic activity and ability to direct human immunodeficiency virus into active transcription units. Here we show that LEDGF promotes the repair of DNA double-strand breaks (DSBs) by the homologous recombination repair pathway. Depletion of LEDGF impairs the recruitment of C-terminal binding protein interacting protein (CtIP) to DNA DSBs and the subsequent CtIP-dependent DNA-end resection. LEDGF is constitutively associated with chromatin through its Pro-Trp-Trp-Pro (PWWP) domain that binds preferentially to epigenetic methyl-lysine histone markers characteristic of active transcription units. LEDGF binds CtIP in a DNA damage-dependent manner, thereby enhancing its tethering to the active chromatin and facilitating its access to DNA DSBs. These data highlight the role of PWWP-domain proteins in DNA repair and provide a molecular explanation for the antiapoptotic and cancer cell survival-activities of LEDGF.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Recombinational DNA Repair/physiology , Transcription Factors/metabolism , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/genetics , Apoptosis , Carrier Proteins/metabolism , Cell Line, Tumor , Cell Survival , Chromatin/metabolism , DNA Breaks, Double-Stranded , Endodeoxyribonucleases , HIV/genetics , HeLa Cells , Humans , Nuclear Proteins/metabolism , RNA Interference , RNA, Small Interfering/genetics , Transcription Factors/antagonists & inhibitors , Transcription Factors/genetics , Virus IntegrationABSTRACT
Maintenance of genome integrity is of critical importance to cells. To identify key regulators of genomic integrity, we screened a human cell line with a kinome small interfering RNA library. WEE1, a major regulator of mitotic entry, and CHK1 were among the genes identified. Both kinases are important negative regulators of CDK1 and -2. Strikingly, WEE1 depletion rapidly induced DNA damage in S phase in newly replicated DNA, which was accompanied by a marked increase in single-stranded DNA. This DNA damage is dependent on CDK1 and -2 as well as the replication proteins MCM2 and CDT1 but not CDC25A. Conversely, DNA damage after CHK1 inhibition is highly dependent on CDC25A. Furthermore, the inferior proliferation of CHK1-depleted cells is improved substantially by codepletion of CDC25A. We conclude that the mitotic kinase WEE1 and CHK1 jointly maintain balanced cellular control of Cdk activity during normal DNA replication, which is crucial to prevent the generation of harmful DNA lesions during replication.
Subject(s)
Cyclin-Dependent Kinases/metabolism , Genomic Instability , S Phase/physiology , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line , Checkpoint Kinase 1 , Cyclin-Dependent Kinases/genetics , DNA Damage , DNA Replication , Flow Cytometry , Genome, Human , Humans , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Kinases/genetics , Protein Kinases/metabolism , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , RNA, Small Interfering/metabolism , cdc25 Phosphatases/genetics , cdc25 Phosphatases/metabolismABSTRACT
The yeast Saccharomyces kluyveri (Lachancea kluyveri), a far relative of Saccharomyces cerevisiae, is not a widely studied organism in the laboratory. However, significant contributions to the understanding of nucleic acid precursors degradation in eukaryotes have been made using this model organism. Here we review eukaryotic pyrimidine degradation with emphasis on the contributions made with S. kluyveri and how this increases our understanding of human disease. Additionally, we discuss the possibilities and limitations of this nonconventional yeast as a laboratory organism.