Repair of DNA-protein crosslinks in mammalian cells

University of Minnesota Ph.D. dissertation. 2018. Major: Pharmacology. Advisor: Colin Campbell. 1 computer file (PDF); 178 pages.The work below describes a new assay called strand-specific primer extension-quantitative polymerase chain reaction (SSPE-qPCR) used to study the repair of DNA-protein crosslinks in mammalian cells. DNA-protein crosslinks (DPCs) are bulky lesions which disrupt important cell processes such as transcription and replication. They are formed by endogenous molecules such as formaldehyde and exogenous damaging agents such as ionizing radiation. However, the repair mechanisms associated with their repair are still unclear. Chapter 1 of this document provides background information on the formation, biological consequences, current models, and methods used to study DPC repair. Chapter 2 describes the SSPE-qPCR assay and its uses/limitations for studying the repair of plasmids containing DPCs or other polymerase-blocking adducts transfected into mammalian cells. Chapter 3 describes results generated using this assay to assess the role of nucleotide excision repair in DPC repair and highlights the versatility of the SSPE-qPCR assay. Chapter 4 extends observations made in Chapter 3 by using SSPE-qPCR to examine repair of DPC-containing plasmids in the presence of a homologous donor. It also provides evidence for homologous recombinational repair of DPCs in mammalian mitochondria. Overall, this work provides additional insight into the mechanisms of DPC repair in the nucleus and mitochondria using a quantitative, flexible assay that has not been available previously

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.publishedVersionPeer reviewe

Metal Reduction by Methobactins

Color poster with text, diagrams, and tables.Methanobactins (mb) are copper-binding peptides or chalkophores secreted by methanotrophs to scavenge copper ions from the environment. A unique feature of these molecules is their ability to reduce and stabilize copper as Cu(I). In the absence of Cu, the molecule will also bind and reduce many other metals including silver, gold and mercury. We have investigated the methanobactin (MB) from Methylosinus trichosporium OB3b, as well as a newly isolated methanobactin from Methylocystis sp. SB2 in order to determine how these novel polypeptides reduce and stabilize Cu(I) and other metals.University of Wisconsin--Eau Claire Office of Research and Sponsored Programs; National Science Foundation-Major Instrumentation gran

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

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