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.