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Rapid, Direct Detection of Bacterial Topoisomerase 1-DNA Adducts by RADAR/ELISA

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Rapid, Direct Detection of Bacterial Topoisomerase 1-DNA Adducts by RADAR/ELISA bioRxiv preprint doi: https://doi.org/10.1101/2020.03.09.984153; this version posted March 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Rapid, direct detection of bacterial Topoisomerase 1-DNA adducts by RADAR/ELISA Devapriya Sinha1,*, Kostantin Kiianitsa1,*, David R. Sherman2, Nancy Maizels1,3 1Department of Immunology, University of Washington, 1959 NE Pacific St., Seattle, WA 98195, USA 2Department of Microbiology, University of Washington, 815 Republican St., Seattle, WA 98102, USA 3Department of Biochemistry, University of Washington, 1959 NE Pacific St., Seattle, WA 98195, USA *The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors. To whom correspondence should be addressed. Tel., +1 206-685-4449; Fax: +1 206- 221-6781; Email: [email protected] Running title: Direct Assay of Bacterial Top1-DNA Adducts Keywords: DNA-protein crosslinK, gyrase, Mycobacteria, tuberculosis, antibiotic, topoisomerase poison 1 Abstract 2 Topoisomerases are proven drug targets, but antibiotics that poison bacterial 3 Topoisomerase 1 (Top1) have yet to be discovered. We have developed a rapid and 4 direct assay for quantification of Top1-DNA adducts that is suitable for high throughput 5 assays. Adducts are recovered by "RADAR fractionation", a quick, convenient 6 approach in which cells are lysed in chaotropic salts and detergent and nucleic acids 7 and covalently bound adducts then precipitated with alcohol. Here we show that 8 RADAR fractionation followed by ELISA immunodetection can quantify adducts formed 9 by wild-type and mutant Top1 derivatives encoded by two different bacterial pathogens, 10 Y. pestis and M. tuberculosis, expressed in E. coli or M. smegmatis, respectively. For 11 both enzymes, quantification of adducts by RADAR/ELISA produces results comparable 12 to the more cumbersome classical approach of CsCl density gradient fractionation. The 13 experiments reported here establish that RADAR/ELISA assay offers a simple way to 14 characterize Top1 mutants and analyze kinetics of adduct formation and repair. They bioRxiv preprint doi: https://doi.org/10.1101/2020.03.09.984153; this version posted March 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 15 also provide a foundation for discovery and optimization of drugs that poison bacterial 16 Top1 using standard high-throughput approaches. 17 18 INTRODUCTION 19 There is a widely recognized need to develop new drugs to treat infections, especially 20 as many microorganisms have developed resistance to antibiotics in common use. 21 Topoisomerases have proven to be effective drug targets not only in infectious disease 22 but also in cancer (1,2). Topoisomerases modulate DNA topology by catalyzing 23 cleavage and then religation of one or both strands of the duplex, forming a covalent 24 topoisomerase-DNA complex as an obligatory reaction intermediate (3) (Fig. 1A). 25 Accumulation of toxic Top1-DNA covalent intermediates contributes to killing of cells in 26 which drugs or mutations impair the normal Top1 reaction cycle. Drugs that target 27 topoisomerases cause the covalent adduct to persist, preventing release of the bound 28 protein and religation of the DNA. This is the mechanism of cell killing by 29 fluoroquinolone antibiotics, such as ciprofloxacin, moxifloxacin, levofloxacin, and 30 ofloxacin, which target DNA gyrase (4); and of chemotherapeutics used to treat cancer, 31 such as topotecan and etoposide (5,6). Mutations in topoisomerases can also impair 32 their ability to release the covalent bond and reseal the DNA duplex. 33 The only bacterial topoisomerases targeted by drugs to date are the Type II 34 topoisomerases, gyrase and topoisomerase IV. Efforts to identify potent small molecule 35 inhibitors of bacterial Type IA enzymes have had only limited success thus far (7). Key 36 to the religation step catalyzed by bacterial Top1 is coordination of a metal ion by 37 aspartate residues of an acidic triad within the DxDxxG motif of the highly conserved 38 Top1 TOPRIM domain (8,9) (Fig. 1B). Mutational analysis has shown that modification 39 of the N- or C-terminal residues of this domain in Top1 from bacterial species, including 40 Escherichia coli, Yersinia pestis, Mycobacterium tuberculosis and Mycobacterium 41 smegmatis, will impair DNA religation, induce an SOS response and cause cell killing 42 (10-14). 43 The absence of a simple, mechanism-based assay that quantifies Top1-DNA 44 adducts formed in vivo has been a stumbling block to drug discovery and development 45 for this target. Some features of topoisomerases themselves contribute to difficulty in 46 systematic detection. Topoisomerase-DNA adducts are normally transient and may 47 resolve spontaneously when drug is removed or limiting; cells may lyse in response to 48 cell killing, releasing adducted complexes into the culture medium. The adducted 49 enzyme may also undergo proteolytic repair, eliminating epitopes for immunodetection, 50 as we recently demonstrated for human endogenous topoisomerase-DNA adducts by 51 an unbiased proteomic approach (15). As the first step toward overcoming these 52 challenges, we have developed a rapid and direct assay for quantification of 53 topoisomerase-DNA adducts formed in living cells. The assay is based on a method 54 referred to as “RADAR” fractionation (16,17), in which cells are lysed in chaotropic salts bioRxiv preprint doi: https://doi.org/10.1101/2020.03.09.984153; this version posted March 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 55 and detergent; nucleic acids and adducted proteins are separated from free protein by 56 alcohol precipitation; and adducted protein is quantified by immunodetection. RADAR 57 fractionation has enabled quantification of adducts formed by bacterial and mammalian 58 topoisomerases (16-23) and by Pol beta (24) and other mammalian proteins (21,25). 59 We have recently validated the ability of RADAR fractionation to enrich adduct-forming 60 proteins from human cells by mass spectrometry (15). RADAR fractionation is cost- 61 and time-effective, and provides a considerable improvement in throughput (100-fold or 62 more) over the classical approach for adduct recovery by CsCl density gradient 63 fractionation (26). 64 Here we describe an assay for bacterial Top1-DNA adducts that combines 65 RADAR fractionation and ELISA assay in microplate format. We have developed this 66 assay using as a model Top1-DNA adducts encoded by two different bacterial 67 pathogens, Y. pestis and M. tuberculosis, expressing epitope-tagged Top1 from 68 inducible constructs. This enables detection by highly specific commercially available 69 antibodies, circumventing the need to identify and characterize antibodies specific to 70 each target of interest. We apply the RADAR/ELISA assay to quantify accumulation of 71 DNA adducts formed by the highly toxic Y. pestis mutant, YpTop1-D117N, expressed in 72 E. coli cells. The Z’ factor of the assay is >0.5, suitable for high throughput applications. 73 We further demonstrate that RADAR lysis disrupts the normally challenging 74 mycobacteria cell wall and enables recovery and quantification of adducts formed by 75 MtTop1-DNA expressed in M. smegmatis. These experiments provide a foundation for 76 discovery and optimization of drugs that poison bacterial Top1 using standard high- 77 throughput approaches. 78 79 MATERIALS AND METHODS 80 81 Cloning and site-directed mutagenesis of MtTop1 constructs 82 83 Gateway LR clonase (Thermo-Fisher) was used to clone the MtTop1 coding sequence 84 from the pENTR vector (pENTR:3646c; (27)) into the pDTNF expression vector 85 (pDTNF:3646c), fusing a FLAG tag to the N-terminal of Top1. The D111A, D111N, and 86 G116S mutants of MtTop1 were generated by QuikChange using Pfu Turbo (Thermo- 87 Fisher). 88 89 Cell culture, Top1 induction, and viability assays 90 91 E. coli strain MG1655 with chromosomally integrated YpTop1 WT (BWYTOP) or 92 YpTop1-D117N (BW117N) bearing an N-terminal TRX tag was a gift from Prof Yuk- 93 Ching Tse-Dinh (Florida International University, USA). Cells were cultured at 37°C in 94 LB medium containing 25 μg/ml chloramphenicol. Top1 expression was induced in bioRxiv preprint doi: https://doi.org/10.1101/2020.03.09.984153; this version posted March 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 95 exponentially growing cultures (OD600=0.3) by addition of arabinose (Sigma Aldrich). 96 Cell division was monitored by measuring OD600. 97 M. smegmatis (mc2155) strain was cultured in 7H9 medium with ADC 98 supplement (HiMedia) as described (28). M. smegmatis was transformed using a 99 MicroPulser™ Electroporator Bio-Rad), and transformed cells selected and propagated 100 in medium containing 200 μg/ml hygromycin B (Thermo-Fisher). MtTop1 expression 101 was induced in exponentially growing (OD600=0.5) cultures of M. smegmatis by addition 102 of 50 ng/ml anhydrotetracycline (ATc; Takara-Clontech) (29). Cell survival was 103 quantified by culturing cells on LB plates containing 200 μg/ml hygromycin B for 3 days 104 at 37°C. 105 M. tuberculosis H37Rv (ATCC 25618) strain was grown in 7H9 medium with 106 ADC supplement (HiMedia) as described (28).
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