The oxidized nucleotide, 8-oxo-7,8-dihydro-2΄-deoxyguanosine (8-oxoG), is one of the most abundant DNA lesions. 8-oxoG plays a major role in tumorigenesis and human disease. Biological consequences of 8-oxoG are mediated in part by its insertion into the genome, making it essential to understand how DNA polymerases handle 8-oxoG. Insertion of 8-oxoG is mutagenic when opposite adenine but not when opposite cytosine. However, either result leads to DNA damage at the primer terminus (3΄-end) during the succeeding insertion event. Extension from DNA damage at primer termini remains poorly understood. Using kinetics and time-lapse crystallography, we evaluated how a model DNA polymerase, human polymerase β, accommodates 8-oxoG at the primer terminus opposite cytosine and adenine. Notably, extension from the mutagenic base pair is favored over the non-mutagenic base pair. When 8-oxoG is at the primer terminus opposite cytosine, DNA centric changes lead to a clash between O8 of 8-oxoG and the phosphate backbone. Changes in the extension reaction resulting from the altered active site provide evidence for a stabilizing interaction between Arg254 and Asp256 that serves an important role during DNA synthesis reactions. These results provide novel insights into the impact of damage at the primer terminus on genomic stability and DNA synthesis.

Valosin-containing protein (VCP), together with several partner proteins, extracts ubiquitinated client proteins from E3 ligase complex and facilitates their degradation through ubiquitin–proteasome system. Therefore, it plays an important role in regulating protein quality control and various cellular pathways. Recent studies also identified VCP as a lineage-specific essential gene in ovarian cancer. An orally bioavailable VCP inhibitor, CB-5083, is currently in Phase I clinical trials because it shows therapeutic effects in multiple tumor xenograft models. However, the mechanism of resistance to CB-5083 is unknown. Here, we characterized molecular mechanism of resistance to CB-5083. Using incremental exposure to CB-5083, we established CB-5083-resistant ovarian cancer cells that showed five- to six-fold resistance in vitro compared with parental cells. Genomic and complementary DNA sequencing of the VCP coding region revealed a pattern of co-selected mutations: (1) missense mutations at codon 470 in one copy resulting in increased ATPase activity and (2) nonsense or frameshift mutations at codon 606 or codon 616 in another copy causing the loss of allele-specific expression. Unbiased molecular docking studies showed codon 470 as a putative binding site for CB-5083. Furthermore, the analysis of somatic mutations in cancer genomes from the Cancer Genome Atlas (TCGA) indicated that codon 616 contains hotspot mutations in VCP. Thus, identification of these mutations associated with in vitro resistance to VCP inhibitors may be useful as potential theranostic markers while screening for patients to enroll in clinical trials. VCP has emerged as a viable therapeutic target for several cancer types, and therefore targeting such hyperactive VCP mutants should aid in improving the therapeutic outcome in cancer patients.

Single-molecule characterization of protein–DNA dynamics provides unprecedented mechanistic details about numerous nuclear processes. Here, we describe a new method that rapidly generates single-molecule information with fluorescently tagged proteins isolated from nuclear extracts of human cells. We demonstrated the wide applicability of this novel technique on undamaged DNA and three forms of DNA damage using seven native DNA repair proteins and two structural variants, including: poly(ADP-ribose) polymerase (PARP1), heterodimeric ultraviolet-damaged DNA-binding protein (UV-DDB), and 8-oxoguanine glycosylase 1 (OGG1). We found that PARP1 binding to DNA nicks is altered by tension, and that UV-DDB did not act as an obligate heterodimer of DDB1 and DDB2 on UV-irradiated DNA. UV-DDB bound to UV photoproducts with an average lifetime of 39 seconds (corrected for photobleaching, τc), whereas binding lifetimes to 8-oxoG adducts were < 1 second. Catalytically inactive OGG1 variant K249Q bound oxidative damage 23-fold longer than WT OGG1, at 47 and 2.0 s, respectively. By measuring three fluorescent colors simultaneously, we also characterized the assembly and disassembly kinetics of UV-DDB and OGG1 complexes on DNA. Hence, the SMADNE technique represents a novel, scalable, and universal method to obtain single-molecule mechanistic insights into key protein–DNA interactions in an environment containing physiologically-relevant nuclear proteins.

Acetaminophen (APAP) overdose is the most common cause of hepatotoxicity and acute liver failure in the United States and many western countries. However, the only clinically approved antidote, N-acetylcysteine, has a limited therapeutic window. 4-Methylpyrazole (4MP) is an antidote for methanol and ethylene glycol poisoning, and we have recently shown that cotreatment of 4MP with APAP effectively prevents toxicity by inhibiting Cyp2E1. To evaluate if 4MP can be used therapeutically, C57BL/6J mice were treated with 300 mg/kg APAP followed by 50 mg/kg 4MP 90 min later (after the metabolism phase). In these experiments, 4MP significantly attenuated liver injury at 3, 6, and 24 h after APAP as shown by 80%-90% reduction in plasma alanine aminotransferase activities and reduced areas of necrosis. 4MP prevented c-Jun c-Jun N-terminal kinase (JNK) activation and its mitochondrial translocation, and reduced mitochondrial oxidant stress and nuclear DNA fragmentation. 4MP also prevented JNK activation in other liver injury models. Molecular docking experiments showed that 4MP can bind to the ATP binding site of JNK. These data suggest that treatment with 4MP after the metabolism phase effectively prevents APAP-induced liver injury in the clinically relevant mouse model in vivo mainly through the inhibition of JNK activation. 4MP, a drug approved for human use, is as effective as N-acetylcysteine or can be even more effective in cases of severe overdoses with prolonged metabolism (600 mg/kg). 4MP acts on alternative therapeutic targets and thus may be a novel approach to treatment of APAP overdose in patients that complements N-acetylcysteine.

The oxidative base damage, 8-oxo-7,8-dihydroguanine (8-oxoG) is a highly mutagenic lesion because replicative DNA polymerases insert adenine (A) opposite 8-oxoG. In mammalian cells, the removal of A incorporated across from 8-oxoG is mediated by the glycosylase MUTYH during base excision repair (BER). After A excision, MUTYH binds avidly to the abasic site and is thus product inhibited. We have previously reported that UV-DDB plays a non-canonical role in BER during the removal of 8-oxoG by 8-oxoG glycosylase, OGG1 and presented preliminary data that UV-DDB can also increase MUTYH activity. In this present study we examine the mechanism of how UV-DDB stimulates MUTYH. Bulk kinetic assays show that UV-DDB can stimulate the turnover rate of MUTYH excision of A across from 8-oxoG by 4–5-fold. Electrophoretic mobility shift assays and atomic force microscopy suggest transient complex formation between MUTYH and UV-DDB, which displaces MUTYH from abasic sites. Using single molecule fluorescence analysis of MUTYH bound to abasic sites, we show that UV-DDB interacts directly with MUTYH and increases the mobility and dissociation rate of MUTYH. UV-DDB decreases MUTYH half-life on abasic sites in DNA from 8800 to 590 seconds. Together these data suggest that UV-DDB facilitates productive turnover of MUTYH at abasic sites during 8-oxoG:A repair.

DNA polymerases are vital for the synthesis of new DNA strands. Since the discovery of DNA polymerase I in Escherichia coli, a diverse library of mammalian DNA polymerases involved in DNA replication, DNA repair, antibody generation, and cell checkpoint signaling has emerged. While the unique functions of these DNA polymerases are differentiated by their association with accessory factors and/or the presence of distinctive catalytic domains, atomic resolution structures of DNA polymerases in complex with their DNA substrates have revealed mechanistic subtleties that contribute to their specialization. In this review, the structure and function of all 15 mammalian DNA polymerases from families B, Y, X, and A will be reviewed and discussed with special emphasis on the insights gleaned from recently published atomic resolution structures.

Somatic mutations are highly enriched at transcription factor (TF) binding sites, with the strongest trend being observed for ultraviolet light (UV)-induced mutations in melanomas. One of the main mechanisms proposed for this hypermutation pattern is the inefficient repair of UV lesions within TF-binding sites, caused by competition between TFs bound to these lesions and the DNA repair proteins that must recognize the lesions to initiate repair. However, TF binding to UV-irradiated DNA is poorly characterized, and it is unclear whether TFs maintain specificity for their DNA sites after UV exposure. We developed UV-Bind, a high-throughput approach to investigate the impact of UV irradiation on protein–DNA binding specificity. We applied UV-Bind to ten TFs from eight structural families, and found that UV lesions significantly altered the DNA-binding preferences of all the TFs tested. The main effect was a decrease in binding specificity, but the precise effects and their magnitude differ across factors. Importantly, we found that despite the overall reduction in DNA-binding specificity in the presence of UV lesions, TFs can still compete with repair proteins for lesion recognition, in a manner consistent with their specificity for UV-irradiated DNA. In addition, for a subset of TFs, we identified a surprising but reproducible effect at certain nonconsensus DNA sequences, where UV irradiation leads to a high increase in the level of TF binding. These changes in DNA-binding specificity after UV irradiation, at both consensus and nonconsensus sites, have important implications for the regulatory and mutagenic roles of TFs in the cell.

DNA is under constant threat of damage from a variety of chemical and physical insults, such as ultraviolet rays produced by sunlight and reactive oxygen species produced during respiration or inflammation. Because damaged DNA, if not repaired, can lead to mutations or cell death, multiple DNA repair pathways have evolved to maintain genome stability. Two repair pathways, nucleotide excision repair (NER) and base excision repair (BER), must sift through large segments of nondamaged nucleotides to detect and remove rare base modifications. Many BER and NER proteins share a common base-flipping mechanism for the detection of modified bases. However, the exact mechanisms by which these repair proteins detect their damaged substrates in the context of cellular chromatin remains unclear. The latest generation of single-molecule techniques, including the DNA tightrope assay, atomic force microscopy, and real-time imaging in cells, now allows for nearly direct visualization of the damage search and detection processes. This review describes several mechanistic commonalities for damage detection that were discovered with these techniques, including a combination of 3-dimensional and linear diffusion for surveying damaged sites within long stretches of DNA. We also discuss important findings that DNA repair proteins within and between pathways cooperate to detect damage. Finally, future technical developments and single-molecule studies are described which will contribute to the growing mechanistic understanding of DNA damage detection.