Bacteria-to-human protein networks reveal origins of endogenous DNA damage

Abstract

52 53 DNA damage provokes mutations and cancer, and results from external carcinogens or 54 endogenous cellular processes. Yet, the intrinsic instigators of DNA damage are poorly 55 understood. Here we identify proteins that promote endogenous DNA damage when 56 overproduced: the DNA-damaging proteins (DDPs). We discover a large network of DDPs 57 in Escherichia coli and deconvolute them into six DNA-damage-causing function clusters, 58 demonstrating DDP mechanisms in three: reactive-oxygen increase by transmembrane 59 transporters, chromosome loss by replisome binding, and replication stalling by transcription 60 factors. Their 284 human homologs are over-represented among known cancer drivers, and their 61 expression in tumors predicts heavy mutagenesis and poor prognosis. Half of tested human 62 homologs, when overproduced in human cells, promote DNA damage and mutation, with DNA-63 damaging mechanisms like those in E. coli. Together, our work reveals DDP networks that 64 provoke endogenous DNA damage and may indicate functions of many human known and newly 65 implicated cancer-promoting proteins. 66 67 68 69 DNA damage often underlies "spontaneous" mutations (Hastings et al.

Transcriptome‐wide association study reveals candidate causal genes for lung cancer

“…12 The human cell line has been authenticated using STR profiling within the last 3 years and all experiments were performed with mycoplasma-free cells. MRC-5V2 (male, SV40-immortalized human lung fibroblasts, Research Resource Identifier (RRID): CVCL_2627, source: Stephen P. Jackson Lab) cell line was maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco, Catalog #: 41965) supplemented with 10% fetal bovine serum (Gibco, Catalog #: 10438034), 2 mM L-glutamine, 100 μg/ml penicillin, and 100 μg/ml streptomycin as previously described.…”

“…MRC-5V2 (male, SV40-immortalized human lung fibroblasts, Research Resource Identifier (RRID): CVCL_2627, source: Stephen P. Jackson Lab) cell line was maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco, Catalog #: 41965) supplemented with 10% fetal bovine serum (Gibco, Catalog #: 10438034), 2 mM L-glutamine, 100 μg/ml penicillin, and 100 μg/ml streptomycin as previously described. 12 Briefly, cells were fixed, permeabilized and stained with γH2AX antibody (Sigma, Catalog #05-636), then samples were measured by a BD LSRFortessa flow cytometer and analyzed using FlowJo software. Gateway compatible AQP3 entry clone was obtained from ccsbBroad gene libraries (ccsbBroadEn_00089).…”

“…Flow-cytometric DNA damage assays and quantification signals were performed as previously described. 12 Briefly, cells were fixed, permeabilized and stained with γH2AX antibody (Sigma, Catalog #05-636), then samples were measured by a BD LSRFortessa flow cytometer and analyzed using FlowJo software. For overproduction experiments, cells with mock transfection were used to set the threshold gating to determine the percentage of GFP − and γH2AX − cells, with 0.5% of control cells gated as the damage threshold as previously validated.…”

Gamblers: an Antibiotic-induced Evolvable Cell Subpopulation Differentiated by Reactive-oxygen-induced General Stress Response

“…Captured images for analysis were chosen randomly. The images were taken with Z-stacks (0.15-μm intervals) and then deconvoluted (DeltaVision SoftWoRx software) to visualize the whole cell for precise and accurate quantification of foci per (Xia et al, 2016; Xia et al, 2018). For each experiment, >400 cells were counted using ImageJ software (NIH) with visual inspection from each independent experiment.…”

“…Cells were grown in the absence or presence of cipro MAC (8.5 ng/mL) to early-, late-log, and stationary phase as for Assays for Ciprofloxacin-induced Mutagenesis (above). The ROS measurement protocol was modified from Gutierrez et al and Xia et al (Gutierrez et al, 2013; Xia et al, 2018). Cells were incubated with ROS-staining dye DHR123 (Invitrogen) for 30 min at 37°C in PBS.…”