Protein acetylation is mediated by histone acetyltransferases (HATs) and deacetylases (HDACs), which influence chromatin dynamics, protein turnover and the DNA damage response. ATM and ATR mediate DNA damage checkpoints by sensing double-strand breaks and single-strand-DNA–RFA nucleofilaments, respectively. However, it is unclear how acetylation modulates the DNA damage response. Here we show that HDAC inhibition/ablation specifically counteracts yeast Mec1 (orthologue of human ATR) activation, double-strand-break processing and single-strand-DNA–RFA nucleofilament formation. Moreover, the recombination protein Sae2 (human CtIP) is acetylated and degraded after HDAC inhibition. Two HDACs, Hda1 and Rpd3, and one HAT, Gcn5, have key roles in these processes. We also find that HDAC inhibition triggers Sae2 degradation by promoting autophagy that affects the DNA damage sensitivity of hda1 and rpd3 mutants. Rapamycin, which stimulates autophagy by inhibiting Tor, also causes Sae2 degradation. We propose that Rpd3, Hda1 and Gcn5 control chromosome stability by coordinating the ATR checkpoint and double-strand-break processing with autophagy.
The RecQ helicases are conserved from bacteria to humans and play a critical role in genome stability. In humans, loss of RecQ gene function is associated with cancer predisposition and/or premature aging. Recent data have shown that the RecQ helicases function during two distinct steps during DNA repair; DNA end resection and resolution of double Holliday junctions (dHJs). RecQ functions in these different processing steps has important implications for its role in repair of double-strand breaks (DSBs) that occur during DNA replication, meiosis and at specific genomic loci such as telomeres.
High-grade epithelial ovarian carcinomas containing mutated BRCA1 or BRCA2 (BRCA1/2) homologous recombination (HR) genes are sensitive to platinum-based chemotherapy and PARP inhibitors (PARPi), while restoration of HR function due to secondary mutations in BRCA1/2 has been recognized as an important resistance mechanism. We sequenced core HR pathway genes in 12 pairs of pretreatment and postprogression tumor biopsy samples collected from patients in ARIEL2 Part 1, a phase II study of the PARPi rucaparib as treatment for platinum-sensitive, relapsed ovarian carcinoma. In 6 of 12 pretreatment biopsies, a truncation mutation in BRCA1, RAD51C, or RAD51D was identified. In five of six paired postprogression biopsies, one or more secondary mutations restored the open reading frame. Four distinct secondary mutations and spatial heterogeneity were observed for RAD51C. In vitro complementation assays and a patient-derived xenograft, as well as predictive molecular modeling, confirmed that resistance to rucaparib was associated with secondary mutations. Significance Analyses of primary and secondary mutations in RAD51C and RAD51D provide evidence for these primary mutations in conferring PARPi sensitivity and secondary mutations as a mechanism of acquired PARPi resistance. PARPi resistance due to secondary mutations underpins the need for early delivery of PARPi therapy and for combination strategies.
The small-subunit (SSU) processome is a large ribonucleoprotein required for the biogenesis of the 18S rRNA and likely corresponds to the terminal knobs visualized by electron microscopy on the 5 end of nascent rRNAs. The original purification of the SSU processome of Saccharomyces cerevisiae resulted in the identification of 28 proteins. Here, we characterize 12 additional protein components, including five small-ribosomalsubunit proteins (Rps4, Rps6, Rps7, Rps9, and Rps14) that had previously been copurified. Our multiple criteria for including a component as a bona fide SSU processome component included coimmunoprecipitation with Mpp10 (an SSU processome component), the U3 snoRNA, and the anticipated pre-rRNAs. Importantly, the association of specific ribosomal proteins with the SSU processome suggests that the SSU processome has roles in both pre-rRNA processing and ribosome assembly. These ribosomal proteins may be analogous to the primary or secondary RNA binding proteins first described in bacterial in vitro ribosome assembly maps. In addition to the ribosomal proteins and based on the same experimental approach, we found seven other proteins (Utp18, Noc4, Utp20, Utp21, Utp22, Emg1, and Krr1) to be bona fide SSU processome proteins.Ribosomes are essential for the translation of mRNA into protein. Ribosome biogenesis in Saccharomyces cerevisiae begins with the transcription of the 35S pre-rRNA, which is then cleaved and processed at more than 10 different processing sites to give rise to the mature 18S, 25S, and 5.8S rRNAs (Fig. 1).Small nucleolar ribonucleoproteins (snoRNPs) are required for many of the different processing steps and modifications that occur relative to the pre-rRNA (16). There are three classes of snoRNPs (H/ACA box, C/D box, and RNase mitochondrial RNA processing) that are required for ribosome biogenesis, each of which contains a small nucleolar RNA (snoRNA). H/ACA box snoRNAs are required for site-specific pseudouridylation of rRNA, while C/D box snoRNAs are required for 2Ј-O-ribose methylation of specific nucleotides in rRNA.The U3 snoRNA and its associated proteins are required for the processing of the small ribosomal subunit at cleavage sites A 0 , A 1 , and A 2 (Fig. 1). Cleavages at A 0 and A 1 in the 5Ј external transcribed spacer mature the 5Ј end of the prerRNA. Cleavage at A 2 or A 3 in internal transcribed spacer 1 separates the small-ribosomal-subunit precursor rRNA from the large-ribosomal-subunit precursors. Defects in cleavage at the A 0 , A 1 , and A 2 sites lead to a reduction in the levels of the 18S rRNA. This reduction causes accumulation of the 35S and 23S pre-rRNAs and a reduction in the levels of the 27SA 2 and 20S pre-rRNAs (39).A large RNP required for the processing of the small-ribosomal-subunit rRNA, called the small-subunit (SSU) processome, has recently been purified (4). This preribosomal complex contains the U3 snoRNA and at least 28 proteins. We defined the SSU processome components as having the following properties. (i) They are nucleolar. (ii) They...
Damage repair mechanisms at transcriptionally active sites during the G0/G1 phase are largely unknown. To elucidate these mechanisms, we introduced genome site-specific oxidative DNA damage and determined the role of transcription in repair factor assembly. We find that KU and NBS1 are recruited to damage sites independent of transcription. However, assembly of RPA1, RAD51C, RAD51, and RAD52 at such sites is strictly governed by active transcription and requires both wild-type Cockayne syndrome protein B (CSB) function and the presence of RNA in the G0/G1 phase. We show that the ATPase activity of CSB is indispensable for loading and binding of the recombination factors. CSB counters radiation-induced DNA damage in both cells and zebrafish models. Taken together, our results have uncovered a novel, RNA-based recombination mechanism by which CSB protects genome stability from strand breaks at transcriptionally active sites and may provide insight into the clinical manifestations of Cockayne syndrome.NA double strand breaks (DSBs) are a most severe type of DNA damage caused by endogenous metabolic processes and exogenous exposure to radiation and chemicals. Unrepaired DSBs induce genomic instability, carcinogenesis, and premature aging. In mammalian cells, DSBs are repaired by either the nonhomologous end joining (NHEJ) or the homologous recombination (HR) pathway. Although it is a common understanding that HR primarily takes place in response to strand breaks in the S-G2 phases of the cell cycle where the undamaged sister chromatids are present as donor templates, recent studies have suggested that homologous pairing also occurs during the G0/G1 phase and is associated with transcription (1), although the mechanisms remain to be elucidated. At active transcription sites, RNA polymerase II (RNA POLII) can bypass base modifications such as 8-oxo guanine but not single strand breaks (SSBs) and DSBs (2-5), indicating that unrepaired strand breaks at transcriptionally active (TA) sites can be especially deleterious and may lead to secondary damage.The Cockayne syndrome B (CSB) gene is defective in approximately two-thirds of patients with Cockayne syndrome (CS), an autosomal recessive disease with diverse clinical signs including severe growth failure, progressive neurodegeneration, and hypersensitivity to sunlight. CSB has an established role in transcription-coupled nucleotide excision repair (TC-NER) of photo lesions. When RNA POLII is stalled at bulky lesions, CSB is loaded to facilitate NER of the transcribed strand (6, 7). As noted, in addition to UV sensitivity, CS patients also manifest severe neurodegeneration (8, 9), suggesting the importance of CS proteins in maintaining genome stability against a broad spectrum of DNA damage. For example, CSB-defective cells are also sensitive to ionizing radiation (IR) (10, 11), which is phenotypically distinctive from classic NER deficiencies and indicates that CSB function is not limited to UV-derived photo lesions. In addition, CSB-deficient mice exhibit a subset of sy...
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