Multiplexed DNA repair assays for multiple lesions and multiple doses via transcription inhibition and transcriptional mutagenesis

Abstract

The capacity to repair different types of DNA damage varies among individuals, making them more or less susceptible to the detrimental health consequences of damage exposures. Current methods for measuring DNA repair capacity (DRC) are relatively labor intensive, often indirect, and usually limited to a single repair pathway. Here, we describe a fluorescence-based multiplex flow-cytometric host cell reactivation assay (FM-HCR) that measures the ability of human cells to repair plasmid reporters, each bearing a different type of DNA damage or different doses of the same type of DNA damage. FM-HCR simultaneously measures repair capacity in any four of the following pathways: nucleotide excision repair, mismatch repair, base excision repair, nonhomologous end joining, homologous recombination, and methylguanine methyltransferase. We show that FM-HCR can measure interindividual DRC differences in a panel of 24 cell lines derived from genetically diverse, apparently healthy individuals, and we show that FM-HCR may be used to identify inhibitors or enhancers of DRC. We further develop a next-generation sequencing-based HCR assay (HCR-Seq) that detects rare transcriptional mutagenesis events due to lesion bypass by RNA polymerase, providing an added dimension to DRC measurements. FM-HCR and HCR-Seq provide powerful tools for exploring relationships among global DRC, disease susceptibility, and optimal treatment.

Mechanism of RNA polymerase II bypass of oxidative cyclopurine DNA lesions

“…The roman numerals (I-V) indicate steps in the bypass. [16][17][18][19]. Prolonged incubation of CydA-stalled TEC10U with 1 mM UTP or ATP followed by a chase with four NTPs generated the bypass runoff product with a rate similar to that observed with CydA-stalled TEC9 (Fig.…”

“…Transcriptional bypass of bulky CPDs in vivo generates mainly WT RNA transcripts, with the polymerase incorporating AMP residues opposite both T residues of the TT dimer (10). A recent report showed an unexpected, error-prone transcriptional CPD bypass in vivo by human Pol II, involving GMP misincorporation opposite the 3′ T-CPD followed by cognate AMP incorporation opposite the 5′ T-CPD (18). Transcriptional CPD bypass in vitro involved nontemplated AMP addition opposite the 3′ T-CPD, resulting in the formation of error-free RNA (6).…”

A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin

“…Depletion of HR proteins in Drosophila cells, but not NHEJ proteins, resulted in retention of IR-induced repair foci inside the heterochromatin domain (Chiolo et al 2011). However, direct determination of all DSB repair pathways used as well as information about the templates used for HR require sequence analysis of repair products (Nagel et al 2014;Soong et al 2015), which is difficult for repetitive DNA. Thus, other pathways, such as NHEJ or single-strand annealing (SSA), could also play an important role in repairing heterochromatic DSBs.…”

Activation of DNA Damage Signaling Components by Diagnostic Computed Tomography (CT) Scans Detected in Patient Samples Using an Electrochemiluminescence-Based Assay Platform

“…These assays include the single cell gel electrophoresis assay (COMET) [33] [34] [35], mass spectrometry based quantification of specific DNA lesions [36], host extract based molecular beacons [37], microarray analysis of transcripts [38] and fluorescence-based multiplex flow cytometric host cell reactivation assays (FM-HCR) that measure the repair of specific lesions [39] [40] [41]. In general, these assays provide valuable data on the integrity of DNA or parts of the DDR, and each assay has proved to be extremely useful for research studies on the cellular DDR and DNA repair capacity.…”

“…High throughput COMET assays, high throughput γ-H2AX foci measures and multiplexed fluorescent host cell reactivation assays have highlighted the value of measuring multiple components or outputs [41] Immunological analysis of epitopes on specific proteins is a proven low-tech approach for analyzing proteins and can be accomplished using immunoblots and enzyme-linked immunosorbent assays (ELISA), and such approaches are the work-horses of most biomedical research and diagnostic labs. Unfortunately, immunoblots and ELISA are not readily applicable to the simultaneous and quantitative analysis of the hundreds of proteins and activated targets associated with the DDR, as they suffer from throughput and dynamic range issues.…”