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Temporal regulation of dUTP biosynthesis limits uracil incorporation during early DNA replication

ABSTRACT: Antimetabolite chemotherapies increase uracil levels in DNA, and thus identification of factors that influence the uracil content in DNA may have implications for understanding uracil-mediated chromosomal instability. We previously showed in the budding yeast Saccharomyces cerevisiae that uracil content in DNA correlates with replication timing, where the earliest and latest replicating regions are depleted in uracil. Here, we manipulated nucleotide biosynthesis enzymes in budding yeast to determine whether the pattern of uracil incorporation could be altered. In strains with high levels of uracil incorporation, deletion of dCMP deaminase (Dcd1) accelerated uracil incorporation at early-firing origins, likely due to rapid dTTP pool depletion. In contrast, increasing the activity of ribonucleotide reductase, which is required for the synthesis of all dNTPs via ribonucleotide diphosphates, lead to dUTP and dTTP pool equilibration and a concomitant increase in uracil content throughout the genome. These data suggest that uracil availability and the dUTP:dTTP ratio are temporally regulated during S phase and govern uracil incorporation into the genome. Therapeutic manipulation of nucleotide biosynthesis in human cells to either increase the dUTP pool or deplete the dTTP pool in early S phase may therefore improve the efficacy of antimetabolite chemotherapies.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE73364 | GEO | 2016/09/23

SECONDARY ACCESSION(S): PRJNA296802

REPOSITORIES: GEO

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Project description:HDAC inhibitors (HDACi) belong to a new group of chemotherapeutics that are increasingly used to treat B-cell-derived malignancies. Such malignancies regularly carry mutational signatures that conform to off-target induction of uracil by the AID/APOBEC family of cytidine deaminases, or downstream processing of uracil. . HDACi suppress thymidylate synthase increasing the cellular dUTP/dTTP ratio and leading to increased pressure on uracil repair machinery due to misincorporated uracil lesions. To investigate potential effect upon other enzymes involved in genomic uracil induction and processing, Jurkat (T-cell lymphoma) and SUDHL5 (B-cell lymphoma) cells were treated with pan-HDACi SAHA prior to SILAC based MS/MS investigation. HDACi treatment mediated significant differential expression of xx and xx proteins in Jurkat and SUDHL5, respectively, and had a substantial impact upon enzymes involved in in pyrimidine metabolism. Surprisingly, uracil N-glycosylase, UNG, was strongly downregulated by HDACi treatment. Further analysis in HEK and HeLa cells revealed that HDACis induce specific loss of the nuclear isoform UNG2 independent of transcription and cell-cycle alterations. More than 80% of UNG2 is degraded proteasomally after 24 hours treatment with SAHA, MS275, Valproate or Na-butyrate, indicating a universal ability of HDACis to mediate loss of UNG2. Targeted MS/MS analysis in HEK cells against a panel of proteins involved in DNA repair, translesion synthesis and nucleotide metabolism, revealed that UNG2 was the most pronounced differentially expressed among these after HDACi treatment. 48 hour treatment lead to a 30-40% increase in uracil lesions in the nuclear genome of HeLa and HEK cells and MS275 treatment in murine CH12F3 cell line mediated robust UNG2-loss accompanied by reduced class switch recombination. Furthermore, our analysis identified the PCNA-associated factor PAF15 among the downregulated proteins. PAF15 is overexpressed in many cancers and suppress TLS by inducing double monoubiquitinylation of PCNA and recruitment of the replicative polymerases. In summary, our findings demonstrate that HDAC inhibition affects the levels of proteins involved in DNA base excision repair, translesion synthesis and pyrimidine metabolism. These findings are important for a wide range of clinical applications of HDACi, such as in rheumatology, HIV-, and cancer treatment.

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Temporal regulation of dUTP biosynthesis limits uracil incorporation during early DNA replication

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