Ubiquitin signaling and the proteasome drive human DNA-protein crosslink repair.

DNA-protein crosslinks (DPCs) are large cytotoxic DNA lesions that form following exposure to chemotherapeutic drugs and environmental chemicals. Nucleotide excision repair (NER) and homologous recombination (HR) promote survival following exposure to DPC-inducing agents. However, it is not known how cells recognize DPC lesions, or what mechanisms selectively target DPC lesions to these respective repair pathways. To address these questions, we examined DPC recognition and repair by transfecting a synthetic DPC lesion comprised of the human oxoguanine glycosylase (OGG1) protein crosslinked to double-stranded M13MP18 into human cells. In wild-type cells, this lesion is efficiently repaired, whereas cells deficient in NER can only repair this lesion if an un-damaged homologous donor is co-transfected. Transfected DPC is subject to rapid K63 polyubiquitination. In NER proficient cells, the DPC is subject to K48 polyubiquitination, and is removed via a proteasome-dependent mechanism. In NER-deficient cells, the DNA-conjugated protein is not subject to K48 polyubiquitination. Instead, the K63 tag remains attached, and is only lost when a homologous donor molecule is present. Taken together, these results support a model in which selective addition of polyubiquitin chains to DNA-crosslinked protein leads to selective recruitment of the proteasome and the cellular NER and recombinational DNA repair machinery.

Cellular Repair of DNA-DNA Cross-Links Induced by 1,2,3,4-Diepoxybutane.

Xenobiotic-induced interstrand DNA-DNA cross-links (ICL) interfere with transcription and replication and can be converted to toxic DNA double strand breaks. In this work, we investigated cellular responses to 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7G-BD) cross-links induced by 1,2,3,4-diepoxybutane (DEB). High pressure liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI⁺-MS/MS) assays were used to quantify the formation and repair of bis-N7G-BD cross-links in wild-type Chinese hamster lung fibroblasts (V79) and the corresponding isogenic clones V-H1 and V-H4, deficient in the XPD and FANCA genes, respectively. Both V-H1 and V-H4 cells exhibited enhanced sensitivity to DEB-induced cell death and elevated bis-N7G-BD cross-links. However, relatively modest increases of bis-N7G-BD adduct levels in V-H4 clones did not correlate with their hypersensitivity to DEB. Further, bis-N7G-BD levels were not elevated in DEB-treated human clones with defects in the XPA or FANCD2 genes. Comet assays and γ-H2AX focus analyses conducted with hamster cells revealed that ICL removal was associated with chromosomal double strand break formation, and that these breaks persisted in V-H4 cells as compared to control cells. Our findings suggest that ICL repair in cells with defects in the Fanconi anemia repair pathway is associated with aberrant re-joining of repair-induced double strand breaks, potentially resulting in lethal chromosome rearrangements.

The 5-Hydroxymethylcytosine Landscape of Prostate Cancer.

Analysis of DNA methylation is a valuable tool to understand disease progression and is increasingly being used to create diagnostic and prognostic clinical biomarkers. While conversion of cytosine to 5-methylcytosine (5mC) commonly results in transcriptional repression, further conversion to 5-hydroxymethylcytosine (5hmC) is associated with transcriptional activation. Here we perform the first study integrating whole-genome 5hmC with DNA, 5mC, and transcriptome sequencing in clinical samples of benign, localized, and advanced prostate cancer. 5hmC is shown to mark activation of cancer drivers and downstream targets. Furthermore, 5hmC sequencing revealed profoundly altered cell states throughout the disease course, characterized by increased proliferation, oncogenic signaling, dedifferentiation, and lineage plasticity to neuroendocrine and gastrointestinal lineages. Finally, 5hmC sequencing of cell-free DNA from patients with metastatic disease proved useful as a prognostic biomarker able to identify an aggressive subtype of prostate cancer using the genes TOP2A and EZH2, previously only detectable by transcriptomic analysis of solid tumor biopsies. Overall, these findings reveal that 5hmC marks epigenomic activation in prostate cancer and identify hallmarks of prostate cancer progression with potential as biomarkers of aggressive disease. SIGNIFICANCE: In prostate cancer, 5-hydroxymethylcytosine delineates oncogene activation and stage-specific cell states and can be analyzed in liquid biopsies to detect cancer phenotypes. See related article by Wu and Attard, p. 3880.

DNA-protein crosslinks are repaired via homologous recombination in mammalian mitochondria.

While mammalian mitochondria are known to possess a robust base excision repair system, direct evidence for the existence of additional mitochondrial DNA repair pathways is elusive. Herein a PCR-based assay was employed to demonstrate that plasmids containing DNA-protein crosslinks are rapidly repaired following electroporation into isolated mammalian mitochondria. Several lines of evidence argue that this repair occurs via homologous recombination. First, DNA-protein crosslinks present on plasmid DNA homologous to the mitochondrial genome were efficiently repaired (21 % repair in three hours), whereas a DNA-protein crosslink present on DNA that lacked homology to the mitochondrial genome remained unrepaired. Second, DNA-protein crosslinks present on plasmid DNA lacking homology to the mitochondrial genome were repaired when they were co-electroporated into mitochondria with an undamaged, homologous plasmid DNA molecule. Third, no repair was observed when DNA-protein crosslink-containing plasmids were electroporated into mitochondria isolated from cells pre-treated with the Rad51 inhibitor B02. These findings suggest that mitochondria utilize homologous recombination to repair endogenous and xenobiotic-induced DNA-protein crosslinks. Consistent with this interpretation, cisplatin-induced mitochondrial DNA-protein crosslinks accumulated to higher levels in cells pre-treated with B02 than in control cisplatin-treated cells. These results represent the first evidence of how spontaneous and xenobiotic-induced DNA-protein crosslinks are removed from mitochondrial DNA.

An integrated functional and clinical genomics approach reveals genes driving aggressive metastatic prostate cancer.

Genomic sequencing of thousands of tumors has revealed many genes associated with specific types of cancer. Similarly, large scale CRISPR functional genomics efforts have mapped genes required for cancer cell proliferation or survival in hundreds of cell lines. Despite this, for specific disease subtypes, such as metastatic prostate cancer, there are likely a number of undiscovered tumor specific driver genes that may represent potential drug targets. To identify such genetic dependencies, we performed genome-scale CRISPRi screens in metastatic prostate cancer models. We then created a pipeline in which we integrated pan-cancer functional genomics data with our metastatic prostate cancer functional and clinical genomics data to identify genes that can drive aggressive prostate cancer phenotypes. Our integrative analysis of these data reveals known prostate cancer specific driver genes, such as AR and HOXB13, as well as a number of top hits that are poorly characterized. In this study we highlight the strength of an integrated clinical and functional genomics pipeline and focus on two top hit genes, KIF4A and WDR62. We demonstrate that both KIF4A and WDR62 drive aggressive prostate cancer phenotypes in vitro and in vivo in multiple models, irrespective of AR-status, and are also associated with poor patient outcome.

The DNA methylation landscape of advanced prostate cancer.

Although DNA methylation is a key regulator of gene expression, the comprehensive methylation landscape of metastatic cancer has never been defined. Through whole-genome bisulfite sequencing paired with deep whole-genome and transcriptome sequencing of 100 castration-resistant prostate metastases, we discovered alterations affecting driver genes that were detectable only with integrated whole-genome approaches. Notably, we observed that 22% of tumors exhibited a novel epigenomic subtype associated with hypermethylation and somatic mutations in TET2, DNMT3B, IDH1 and BRAF. We also identified intergenic regions where methylation is associated with RNA expression of the oncogenic driver genes AR, MYC and ERG. Finally, we showed that differential methylation during progression preferentially occurs at somatic mutational hotspots and putative regulatory regions. This study is a large integrated study of whole-genome, whole-methylome and whole-transcriptome sequencing in metastatic cancer that provides a comprehensive overview of the important regulatory role of methylation in metastatic castration-resistant prostate cancer.

A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells.

The purpose of this method is to provide a flexible, rapid, and quantitative technique to examine the kinetics of DNA-protein crosslink (DPC) repair in mammalian cell lines. Rather than globally assaying removal of xenobiotic-induced or spontaneous chromosomal DPC removal, this assay examines the repair of a homogeneous, chemically defined lesion specifically introduced at one site within a plasmid DNA substrate. Importantly, this approach avoids the use of radioactive materials and is not dependent on expensive or highly-specialized technology. Instead, it relies on standard recombinant DNA procedures and widely available real-time, quantitative polymerase chain reaction (qPCR) instrumentation. Given the inherent flexibility of the strategy utilized, the size of the crosslinked protein, as well as the nature of the chemical linkage and the precise DNA sequence context of the attachment site can be varied to address the respective contributions of these parameters to the overall efficiency of DPC repair. Using this method, plasmids containing a site-specific DPC were transfected into cells and low molecular weight DNA recovered at various times post-transfection. Recovered DNA is then subjected to strand-specific primer extension (SSPE) using a primer complementary to the damaged strand of the plasmid. Since the DPC lesion blocks Taq DNA polymerase, the ratio of repaired to un-repaired DNA can be quantitatively assessed using qPCR. Cycle threshold (CT) values are used to calculate percent repair at various time points in the respective cell lines. This SSPE-qPCR method can also be used to quantitatively assess the repair kinetics of any DNA adduct that blocks Taq polymerase.

A quantitative PCR-based assay reveals that nucleotide excision repair plays a predominant role in the removal of DNA-protein crosslinks from plasmids transfected into mammalian cells.

DNA-protein crosslinks (DPCs) are complex DNA lesions that induce mutagenesis and cell death. DPCs are created by common antitumor drugs, reactive oxygen species, and endogenous aldehydes. Since these agents create other types of DNA damage in addition to DPCs, identification of the mechanisms of DPC repair is challenging. In this study, we created plasmid substrates containing site-specific DPC lesions, as well as plasmids harboring lesions that are selectively repaired by the base excision or nucleotide excision repair (NER) pathways. These substrates were transfected into mammalian cells and a quantitative real-time PCR assay employed to study their repair. This assay revealed that DPC lesions were rapidly repaired in wild-type human and Chinese hamster derived cells, as were plasmids harboring an oxoguanine residue (base excision repair substrate) or cholesterol lesion (NER substrate). Interestingly, the DPC substrate was repaired in human cells nearly three times as efficiently as in Chinese hamster cells (>75% vs ∼25% repair at 8 h post-transfection), while there was no significant species-specific difference in the efficiency with which the cholesterol lesion was repaired (∼60% repair). Experiments revealed that both human and hamster cells deficient in NER due to mutations in the xeroderma pigmentosum A or D genes were five to ten-fold less able to repair the cholesterol and DPC lesions than were wild-type control clones, and that both the global genome and transcription-coupled sub-pathways of NER were capable of repairing DPCs. In addition, analyses using this PCR-based assay revealed that a 4 kDa peptide DNA crosslink was repaired nearly twice as efficiently as was a ∼38 kDa DPC, suggesting that proteolytic degradation of crosslinked proteins occurs during DPC repair. These results highlight the utility of this PCR-based assay to study DNA repair and indicate that the NER machinery rapidly and efficiently repairs plasmid DPC lesions in mammalian cells.

Spectral and copper binding properties of methanobactin from the facultative methanotroph Methylocystis strain SB2.

Methanobactin (mb) is the first characterized example of a chalkophore, a class of copper-binding chromopeptides similar to iron-binding siderophores. Structural, redox, themodynamic, and spectral studies on chalkophores have focused almost exclusively on the mb from Methylosinus trichosporium OB3b (mb-OB3b). The structural characterization of a second mb from Methylocystis strain SB2 (mb-SB2) provides a means to examine the core structural features and metal binding properties of this group of chromopeptides. With the exception of the 5-membered rings (either oxazolone or imidazolone), enethiol groups, and the N-terminus oxo group, the structure of mb-SB2 differs markedly from mb-OB3b. In particular the amino acids commonly associated with metal coordination and redox activity found in mb-OB3b, Cys, Met, and Try, are replaced by Ala or are missing in mb-SB2. In this report the spectral and thermodynamic properties of mb-SB2 are presented and compared to mb-OB3b. The results demonstrate that the spectral and basic copper binding properties of both methanobactins are similar and the unique copper binding capacity of both methanobactins lies primarily in the pair of five-membered rings and associated enethiol groups. The remaining portions of the methanobactin appear to provide the scaffolding that brings together of the two ring systems to produce the tetrahedral binding site for copper binding.