BRCA1 plays a key role in homologous recombination (HR) DNA repair. Accordingly, changes that downregulate BRCA1, including BRCA1 mutations and reduced BRCA1 transcription, due to promoter hypermethylation or loss of the BRCA1 transcriptional regulator CDK12, disrupt HR in multiple cancers. In addition, BRCA1 has also been implicated in the regulation of metabolism. Here, we show that reducing BRCA1 expression, either by CDK12 or BRCA1 depletion, led to metabolic reprogramming of ovarian cancer cells, causing decreased mitochondrial respiration and reduced ATP levels. BRCA1 depletion drove this reprogramming by upregulating nicotinamide N-methyltransferase (NNMT). Notably, the metabolic alterations caused by BRCA1 depletion and NNMT upregulation sensitized ovarian cancer cells to agents that inhibit mitochondrial metabolism (VLX600 and tigecycline) and to agents that inhibit glucose import (WZB117). These observations suggest that inhibition of energy metabolism may be a potential strategy to selectively target BRCA1-deficient high-grade serous ovarian cancer, which is characterized by frequent BRCA1 loss and NNMT overexpression.Significance: Loss of BRCA1 reprograms metabolism, creating a therapeutically targetable vulnerability in ovarian cancer.
Substitution rates in plant mitochondrial genes are extremely low, indicating strong selective pressure as well as efficient repair. Plant mitochondria possess base excision repair pathways; however, many repair pathways such as nucleotide excision repair and mismatch repair appear to be absent. In the absence of these pathways, many DNA lesions must be repaired by a different mechanism. To test the hypothesis that double-strand break repair (DSBR) is that mechanism, we maintained independent self-crossing lineages of plants deficient in uracil-N-glycosylase (UNG) for 11 generations to determine the repair outcomes when that pathway is missing. Surprisingly, no single nucleotide polymorphisms (SNPs) were fixed in any line in generation 11. The pattern of heteroplasmic SNPs was also unaltered through 11 generations. When the rate of cytosine deamination was increased by mitochondrial expression of the cytosine deaminase APOBEC3G, there was an increase in heteroplasmic SNPs but only in mature leaves. Clearly, DNA maintenance in reproductive meristem mitochondria is very effective in the absence of UNG while mitochondrial genomes in differentiated tissue are maintained through a different mechanism or not at all. Several genes involved in DSBR are upregulated in the absence of UNG, indicating that double-strand break repair is a general system of repair in plant mitochondria. It is important to note that the developmental stage of tissues is critically important for these types of experiments.
Current mitochondrial purification techniques are tedious and protracted due to their emphasis on recovering physiologically active mitochondria. However, for studies that are exclusively interested in isolating mitochondrial DNA (mtDNA) for applications such as PCR and sequencing, respiring mitochondria À and the complex procedures that stem from the need to retain their function À are unnecessary. Still, global DNA extraction methods have proven insufficient for mitochondrial DNA isolation because nuclear mitochondrial DNA segments (NUMTs) pose unique challenges to accurate mtDNA quantification and characterization. We present a rapid and simple extraction technique that maximizes recovery of mitochondrial DNA from plant cells, while minimizing the presence of nuclear DNA. Through realtime PCR, we show that this method provides a significant increase in the enrichment of mitochondrial DNA compared to that of nuclear DNA in both Arabidopsis thaliana and Brassica rapa. This method has important implications for future mitochondrial DNA analyses as it possesses few procedural limitations and minimizes the analytical problems typically associated with mtDNA purification by other techniques. ARTICLE HISTORY
<p>Supplementary Figures S1-6. Supplementary Fig. S1 shows that CDK12 depletion alters ATP and ADP levels without affecting glycolysis in cell lines. Supplementary Fig. S2 shows that BRCA1 depletion does not affect glycolysis, that CDK12 and BRCA1 depletion reduce ATP levels in ex vivo HGSOC PDX cultures, that transient BRCA1 expression increase OCR in BRCA1-deficient cells, that CDK12 and BRCA1 depletion do not affect mitochondrial DNA levels, and levels of BRCA1 and CDK12 mRNA expression in cells transfected with CDK12 siRNAs and BRCA1 expression plasmid. Supplementary Fig. S3 shows BRCA2 and RAD51 depletion do not affect OCR but do sensitize to olaparib. Supplementary Fig. S4 shows a map of known BRCA1 transcripts, the exons that are targeted by the BRCA1 siRNAs used in this study, primers used to assess expression of alternative BRCA1 transcripts, NNMT mRNA expression in cells transfected with BRCA1 siRNAs, and binding of BRCA1 to the NNMT promoter using ChIP. Supplementary S5 shows that BRCA2 and RAD51 depletion do not sensitize to VLX600 but do sensitize to olaparib. Supplementary Fig. S6 shows that CDK12 and BRCA1 depletion does not further suppress ATP levels in NNMT-overexpressing cells.</p>
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