STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

[0001] This invention was made with government support under grant numbers

NIH/NIGMS GM107333 awarded by the National Institutes of Health/ National Institute of General Medical Sciences. The government has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS AND INCORPORATION BY

REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0002] This application claims priority to U.S. Provisional Patent Application No.

62/402,605, filed September 30, 2016, the disclosure of which is hereby incorporated by reference in its entirety.

[0003] This application contains, as a separate part of the disclosure, a Sequence Listing in computer-readable form which is incorporated by reference in its entirety and identified as follows: Filename: 51251_Seqlisting.txt; Size 643 bytes; Created: September 29, 2017.

FIELD OF INVENTION

[0004] The disclosure relates generally to materials and methods for detecting DNA mutagenesis.

BACKGROUND

[0005] Genomic mutation is the direct cause of cancer, amyotrophic lateral sclerosis (ALS), haemophilia, neurofibromatosis, and many other diseases. It is also the driving force of evolution. The frequency of genomic mutation is affected by many environmental factors including chemicals, radiation, and cellular oxidants. Multiple mechanisms exist to regulate and control the effects of those environmental factors in order to keep the rate of genetic mutation at a low level. Failure of any of those mechanisms may lead to diseases.

[0006] As a common response to changing environmental cues, post-translational modifications (PTM) of proteins can promptly modulate cellular functions without de novo translation or transcription. Among them, arginylation is the post-translational addition of an extra arginine to an existing peptide chain, usually to the N-terminus, thus capable of changing the surface charge as well as the primary sequence of the target [1]. Arginylation is mediated by arginyltransferase 1 (Atel) [2, 3], an evolutionarily conserved enzyme found in eukaryotic organisms and some bacteria [4-6] . Nearly a hundred proteins have been found to be arginylated and the list of identified substrates is continuously growing [7, 8], suggesting a widespread effect of this PTM in vivo.

[0007] Mounting evidence has suggested an involvement of arginylation in stress response, a naturally occurring process undertaken by cells in stressing conditions, which often lead to growth-arrest or cell death. For more than two decades, researchers have repeatedly observed signs of increased arginylation activity in tissues of animals (such as rat, chicken, and fish) following a variety of insults including nerve crush injury or whole body hyperthermia [9- 18]. At the molecular level, arginylation is often observed on proteins that are nitrosylated, oxidized, or misfolded, which are common consequences of stress [19-21]. These evidence indicate a role for arginylation in physiological response to environmental stress, and therefore also indicate that arginylation is involved in the regulation of DNA mutagenesis. However, the exact nature of arginylation' s involvement in these processes has been unclear.

[0008] Arginylation detection using previous methods is difficult, which hinders progress of the field. Previous methods includes sequencing by Mass Spectrometry or by chemical methods such as Edman degradation, and labeling of the target protein with radioactively arginine. Previous methods rely on expensive instruments with limited access or hazardous materials requiring regulatory supervision. Arginylation can be detected by an antibody, which does not require the hazardous chemicals and expensive machinery required by previous methods. However, ELISA-based assays wherein an antibody specifically recognizing the arginylated form of a short peptide have significant disadvantages. Such an assay can be used to test arginylation of purified (ex vivo) proteins including the Atel enzyme [53]. However, this format is poorly compatible for measuring arginylation activity in the cell or in cell lysates, which contains many endogenous arginylated proteins that cross-react with the antibody in the ELISA assay. Furthermore, arginylation can often lead to substrate degradation. The short peptides used in previous methods cannot be reliably quantified without a complex method and high-precision instrument, therefore limiting its ability to accurately measure arginylation activity. The disclosure provides a method for, e.g., directly evaluating Atel -mediated arginylation inside the cell or in a cell lysate.

[0009] Measuring and comparing mutation frequency at the single cell level is an important task for research related to cancer and many other diseases. Previous methods developed to quantitatively examine mutation frequency in single cells have limitations. One method utilizes endogenous metabolic enzymes that are either required to detoxify certain toxic chemicals or to generate an essential metabolite. The principle of this type of assay is that a loss-of-function mutation of those genes renders a cell vulnerable to toxic chemicals or make certain metabolites unavailable. Examples of this type of method include assays based on Hypoxanthine Phosphoribosyltransferase (HPRT) gene. However, detoxification of toxic chemicals or synthesis of metabolites often can be carried out by alternative pathways.

Therefore, the outcome of previous enzyme-based assays is heavily dependent on the cell type, making this assay unsuitable for comparing mutation frequencies across different cell lines. Also, the treatment of cells with toxic chemicals or nutrient-deprivation creates cellular stress, which is anticipated to affect mutation frequency, therefore creating further complexity and ambiguity for interpreting results.

[0010] Another type of assay uses a single fluorescent or luminescent protein as reporter, which can be examined with optical instruments such as flow cytometers, microscopes, and plate readers. The principle of this type of assay is that if a mutation enables (on) or disables (off) the light-rendering property of the reporter protein, it can be detected by an optical instrument. While this method requires no toxic chemicals or nutrient-deprivation, the method still suffers from unreliability for failure to distinguish "on'V'off" signals arising from other non-mutation related events, such as a loss of reporter vectors during transient expression, uneven transfection and/or incorporation rates among different cell types, or changes in transcription and translation for the individual reporter gene.

[0011] As explained herein, previous methods of detecting arginylation and measuring and comparing mutation frequency at the single cell level suffer from disadvantages that render the methods unreliable or unworkable in a wide array of settings. The disclosure provides improved materials and methods for addressing the need in the field.

SUMMARY OF INVENTION

[0012] The disclosure provides a method for measuring the activity of global arginylation activity, e.g., inside an intact cell, in cell lysates, or in a reconstituted solution with purified proteins. It also provides a method suitable for accurately detecting DNA mutagenesis frequency with a single cell resolution for ratio that is lower than 1 per million.

[0013] Arginylation activity is globally down-regulated by the deletion or silencing of the ATE1 gene, leading to reduction in cellular sensitivities to a variety of stressing factors, resulting in bypass of growth-arrest or reduction of cell death under stress. In addition, the Atel protein level and the global arginylation activity are increased in cells under stress, and Atel mediates cell death in an arginylation-dependent manner. Atel is needed for suppressing the outcome of DNA mutagenesis during DNA-damaging stress. This disclosure describes an example of a post-translational modification having a global effect on DNA mutagenesis.

[0014] Disclosed herein are methods for, e.g., detecting arginylation activity using a recombinant reporter fusion protein. One such method includes introducing into a cell an expression vector comprising a nucleic acid sequence encoding a reporter protein fused to a substrate for arginyltransferase 1 (Atel), such that a reporter fusion protein comprising the reporter protein and Atel substrate is produced. The method further comprises measuring arginylation of the substrate and the reporter protein. The ratio of arginylated substrate to reporter protein is calculated. The ratio is useful for, e.g., characterizing the levels of arginylation. While not wishing to be bound by any particular theory, an increase in arginylation in the cell corresponds to increased cell stress. In some embodiments, arginylation is detected using an arginylation-specific antibody. In some embodiments, the reporter protein is green fluorescent protein (GFP).

[0015] In various embodiments, the arginylation substrate is fused to a cleavable ubiquitin domain located N-terminal to the arginylation substrate. In related embodiments, the ubiquitin domain is cleaved by ubiquitin hydrolase.

[0016] Further disclosed herein is a method for, e.g., estimating DNA mutation frequency (optionally at single-cell resolution) using a reporter plasmid. One such method comprises (a) introducing into a cell a reporter plasmid comprising nucleic acids encoding a promoter, a first reporter protein, an internal ribosome entry site (IRES), and a second reporter protein. The first reporter protein and the second reporter protein are different proteins (i.e., the first and second reporter proteins are not both green fluorescent protein). Either the nucleic acid encoding the first reporter protein comprises a premature stop codon and mutation of the stop codon (e.g., to a sense codon) results in the expression of the full-length first reporter protein, or the nucleic acid encoding the second reporter protein comprises a premature stop codon and mutation of the stop codon results in the expression of the full-length second reporter protein. The method further comprises measuring the first reporter protein and the second reporter protein, and calculating the ratio of first reporter protein to second reporter protein. The ratio allows characterization of the level of mutagenesis of the stop codon within the first or second reporter protein coding sequence, and is indicative of levels of DNA mutagenesis within the cell. In this regard, the reporter protein sequence lacking the premature stop codon serves as an internal control for transcription and translation. In some embodiments, first or second reporter protein is a fluorescent protein; optionally, both the first reporter protein and the second reporter protein are fluorescent proteins. In related embodiments, the reporter proteins are green fluorescent protein (GFP) and red fluorescent protein (RFP). The technique has a resolution of single cell by nature. The assay results can be detected using instruments with single-cell resolution such as a flow cytometer or an optical microscope to examine the number of cells with the first and the second reporter protein. As such, the method's detection limit is, in various embodiments, better than 1 per million with a false- positive rate lower than 5%, as illustrated in Figure 8.

[0017] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patents, and patent applications mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the particular embodiments discussed below are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF DRAWINGS

[0018] Figures 1A-1F. Knockout or knock-down of ATEl decreases cellular sensitivity towards stressing conditions. A) Growth test using serial dilutions of wild-type (WT) and atelA yeast (S. cerevisae, BY4741 strain, unless otherwise mentioned) in non-stressing condition or in the presence of one of the following stressors: common cellular oxidant

(H ₂ 0 ₂ , ImM), heavy metal (CdCl ₂ , 200μΜ), high salt (NaCl, 1M), or high temperature

(40°C). For H ₂ 0 ₂ treatment, cells were plated on SD-plates with glucose. For the other treatments and the non- stressing controls, cells were plated on YPD-plates. B) Growth curves of WT and atelA yeast cultured in liquid media, in the presence (right graph, 0.5mM H ₂ 0 ₂ ) or absence (non-stress condition, left graph) of the oxidative stressor H ₂ 0 ₂ . Error bars represents standard deviation (SD), n=3. C) Growth test using WT or atelA yeast (strain

W303) in non-stressed conditions or in the presence of the heavy metal CdCl ₂ (300μΜ). D)

Viabilities of WT or ATEl -KO mouse embryonic fibroblasts (MEF) after 12 hours treatments with increasing concentrations of cellular oxidant H ₂ 0 ₂ (left graph), bacterial toxin STS (middle graph), or heavy metal CdCl ₂ (right graph). The number of viable cells after H ₂ 0 ₂ treatment was measured by Calcein AM, a cellular dye that emits fluorescence only in live cells. The number of viable cells after STS and Cdcl ₂ treatments was directly counted with an automated cell counter (TC-20 from Biorad) with the cross-staining of Trypan Blue, which stains dead cell but not live cells. In each type of cell, one sample of cells with control treatment (DMSO only) was used as normalization for other samples, for WT (left bar) or ATE1-KO (right bar), respectively. Error bar represents standard error of mean (SEM) of multiple repeats (n=4 for H ₂ 0 ₂ and STS, and n=3 for Cdcl ₂ ). E) Left panel, immunoblot analysis of the steady- state levels of mammalian Atel (mAtel) in MEF transfected with either shRNA specific against mouse ATE1 (sh-mArEi) or shRNA against GFP (used as a non-targeting control). The band of mAtel was probed with a monoclonal antibody

(Millipore, clone #6F11) recognizing all four major splicing variants of Atel in mammalian cells, which are almost identical in molecular sizes and poorly understood in functional differences. Actin was used as a loading control. The right-panel graph shows the

quantification of viability of MEF transfected with Atel knockdown (ShRNAmate, right bar) or non-targeting control (ns, left bar), after treatment with H ₂ 0 ₂ for 12 hours, as measured by the cellular viability indicator Calcein AM. Error bars represent SEM (n=4). F) Similar to E, except that human foreskin fibroblast (HFF) and shRNA against human ATE1 (sh-hATEl) were used. The left panel shows the Western blot. The right-panel graph shows the viability of HFF after treatment of H ₂ 0 ₂ (untreated, left bar; shGFP control, middle bar; sh-hATEl, right bar on graph) Error bars represent SEM (n=4). Throughout this study, the /?-value was calculated by two-tailed Student's t-test between specific data points, unless otherwise indicated.

[0019] Figures 2A-2F. Knockout ofATEl in yeast relieves growth-arrest and suppresses cell death during stress response. A) Viability of WT or atel I. yeast after 3 hours treatments with the indicated concentrations of the oxidative stressor H ₂ 0 ₂ , measured by the colony- forming unit assay and normalized to non-stressed conditions. B) On the left are

representative microscopic images showing the results of the TUNEL assay, which specifically detects DNA fragments generated during late stage of apoptosis. The WT and atel A yeast treated with H ₂ 0 ₂ 0.5mM. DIC images show the number of cells while the fluorescent images show cells with positive apoptosis signals (fluorescein). The graph in the right panel represents the percentage of apoptotic cells determined by TUNEL assay in WT (left bar) and atelA yeast (right bar) treated with increased concentrations of H ₂ O ₂ . C) Growth curves of WT and atelA yeast cultured in liquid media, in the presence or absence of the heavy metal stressor CdCl ₂ of 150μΜ. Error bars represents SEM (n=3). D) Viability of WT or atelA yeast after indicated hours of treatments with 150μΜ CdCl ₂ , measured by the colony-forming unit assay and normalized to cells at time 0 (before the application of stressor). Error bars represents SEM (n=3). E) Growth test using serial dilutions of wild-type (WT) and atelA yeast, either in non-stressing room temperature (RT), or in high-temperature (40°C) for 48 hours followed by recovery at RT for another 48 hours. After recovery at RT, the numbers of colonies emerged in the lowest dilution (xlO ) were quantified. The corresponding numbers for yeast constantly cultured at RT was used for normalization for WT and atelA yeast separately. Left panel, images of the plates, right panel graph showing the quantification of the colony formation ability after recovering at R/T. Error bars represent SEM (n=4 for WT (left bar), n=3 for <2ieiA(right bar). F) Growth test using serial dilutions of wild-type (WT) and atelA yeast, either in non-stressing room temperature (RT), or in high-temperature (42°C) for 48 hours followed by recovery at RT for another 72 hours.

[0020] Figures 3A-3B. Knockout of ATE1 in MEF results in attenuated apoptosis and growth-arrest during stress response. A) Annexin-V-Alexa Fluor™ 488 (from Thermo Fisher) was used to detect phosphatidylserine inversion on the peripheral membrane in an early stage of apoptosis. WT or ATE1-KO MEF were treated with STS or CdCl ₂ of different concentrations for 5 hours. Propidium iodide (PI) was used to label necrotic and late apoptotic fractions. The cell population is analyzed by FACS. On the left panel representative FACS charts are shown. The gate settings for Annexin-V- and Pi-detection are indicated. Quantification of data from three independent repeats (n=3) are shown in graphs presented on the right side. ATE1 -KO cells have a much lower apoptotic rate than WT cells. No obvious difference in Pi-staining was found in the conditions tested. B) On the left panel are representative microscopic images showing WT and ATE1 -KO MEF treated with H ₂ 0 ₂ 0.5mM. EdU (red fluorescently labeled), which is incorporated into newly synthesized DNA during the S phase of cell cycle, was used as a marker for cell proliferation. Hoechst 33342 (blue) was used to show the number and morphology of nucleus. Only cells with normal nuclear morphology and no sign of apoptotic bodies were included for quantification. At least 10 randomly chosen images in each group were used to generate the graphs shown on the right side for quantification of active replicating cells with EdU incorporated into their nucleus, in the presence of stressors H ₂ O ₂ and CdCl ₂ . For the bar graphs, MEF-WT (shown in left bar), ATE1 -KO (shown right bar). Error bars represents SEM.

[0021] Figures 4A-4F. The levels of Atel protein and global arginylation activity are up- regulated during stress. A) A scheme illustrating how DD- 15-GFP is used as the reporter of arginylation activity. The fusion protein containing a stretch of 15 amino acids starting with two aspartic acids (D) derived from the N-terminus of mammalian beta-actin, a known substrate of arginylation [31]. This peptide is fused with an N-terminal ubiquitin, which is cleaved co-translationally by endogenous de-ubiquitylation enzymes in eukaryotic system and leaves the aspartic acids as the new N-terminus. The arginylation state of this reporter can be probed with an anti-RDD antibody, which only reacts with the arginylated form of the reporter protein. A C-terminal GFP tag is used to facilitate the detection of steady state level of the reporter protein by immunoblotting with anti-GFP antibody. B) The arginylation level of DD- 15-GFP expressed in either WT or atel A yeast was examined with anti-RDD antibody, which only shows a visible signal in the WT cells. An antibody for GFP was used to probe the total protein level of the DD- 15-GFP. PGK was used as a loading control for total yeast cellular proteins. C) Illustrative scheme (left panel) and representative

immunoblots (right panel) showing the arginylation activity in cell lysates prepared from yeast exposed to 1M NaCl stress for increasing times. The lysates were then mixed with the recombinant protein DD- 15-GFP prepared from atel A yeast for an in-lysate arginylation reaction for 10 minutes at RT. The arginylation level of the reporter protein was detected by immunoblotting with anti-RDD antibody. The steady-state level of the reporter protein was probed with anti-GFP. An anti-3-phosphoglycerate kinase (PGK) antibody was also used as loading control. D) On the left, a scheme illustrating the domain structure of the "in locus" GFP-fused Atel, which is driven be the endogenous ATE1 promoter at the native

chromosome locus (Chromosome VII) in the yeast (termed "endo: Atel-GFP"). The right panels present immunoblots showing the steady-state levels of "endo: Atel-GFP" in yeast treated with increasing concentrations of different stressors: H ₂ 0 ₂ (left) or NaCl (right). Tubulin or PGK were used as loading controls. E) WT MEF were exposed to increased concentrations of H ₂ 0 ₂ for 30 hours. The lysates from all these cells, as well as untreated ATEl-KO MEF (as a control), were then mixed with the recombinant protein DD- 15-GFP purified from atel A yeast for an in-lysate arginylation reaction for 45 minutes at RT. The arginylation level of the reporter protein was detected by immunoblotting with anti-RDD antibody. The steady-state level of the reporter protein was probed with anti-GFP. Actin antibody was used as loading control. The graph on the right side shows quantification from 4 independent repeats. F) Left: representative immunoblots showing the levels of endogenous Atel proteins in MEF treated with increasing concentrations of Η ₂ 0 ₂ for 30 hours, detected by a specific antibody for mouse Atel (from Millipore, clone 6F11) recognizing all four major Atel splicing variants. Actin was used as loading controls. Right: quantification of the endogenous Atel level in MEF treated with H ₂ 0 ₂ from 3 independent repeats. The Atel level was calculated by normalization with actin loading control, and then further normalized to the level at non-stressing condition (0 μΜ H ₂ 0 ₂ ). In all above figures, error bars represent SEM and statistical significance was calculated by Student's t-test.

[0022] Figures 5A-5E. The increase of Atel triggers cell death in yeast in a manner that is dependent on its arginylation activity. A) The scheme in the top panel shows the domain structure of plasmid pGALl: ATE1, in which the coding sequence of recombinant protein is preceded by the inducible GAL1 promoter. The picture in the bottom panel shows the growth of atel I. yeast cells carrying either the empty expression vector or pGALl: ATE1 by a serial dilution growth assay on either plate containing glucose (suppressing) or galactose

(inducing). B) Graph showing the viabilities of atel I. yeast cells carrying either the empty expression vector or pGALl: ATE1 in different time points following the initiation of galactose-induced expression, as measured by the numbers of colony-forming cells per OD unit (CFU) that were normalized to starting data point time 0, for Atel or vector control separately. Error bar represents SEM (n>=3). C) The top scheme shows the domain structure of a recombinant Atel fused with a linker and a C-terminal GFP, driven by the pGALl promoter, termed "/?GAL: Atel-GFP". Immunoblot analysis of the steady-state levels of wild- type and C20,23S mutant Atel after 9-hour culture in the presence of non-inducing (glucose) or inducing (galactose) carbon sources. PGK was used as loading controls. Anti-GFP was used to probe the steady-state levels of the recombinant "/?GAL: Atel-GFP" (WT or mutant). D) Left panel showing the procedure of using an in-lysate arginylation assay to measure the activities of recombinant Atel-GFP, either the WT version or the C20,23S mutant, which were expressed for 9 hours in atel IS. yeast (See Fig. 5C for the steady state levels of expressed proteins). Anti-RDD was used to indicate the level of arginylated reporter. Anti-GFP was used to show the total amount of reporter protein (DD- 15-GFP) in each sample, as well as the total amount of Atel-GFP (either WT or mutant) present in each sample. These two bands were distinguished by their difference in molecular weight (27kD vs. 92kD). E) Representative pictures of growth test using serial dilutions of atelA yeast carrying either the empty expression vector or pGALl -Ate 1-GFP, or pGALl - Ate 1 -C20,23 -S -GFP, in media containing non-inducing (glucose) or inducing (galactose) carbon sources.

[0023] Figures 6A-6D. Mammalian Atel is required for cellular sensitivity to stressors in a manner dependent on its arginylation activity. A) Representative immunoblots showing the levels of Atel protein detected by a specific mouse Atel antibody (from Millipore, clone 6F11) as either endogenous Atel in WT MEF, or the recombinant mouse Atel-isofrom 1 (WT or C23-26S mutant) fused with a C-terminal GFP stably transfected in ATE1-KO cells. Actin was used as a loading control. B) Left panel showing the procedure of using an in- lysate arginylation assay to measure the activities of either the WT version of mAtel.l-GFP or the mutant with cysteine 23 and 26 to serine replacement (referred as mAtel.l-mut-GFP in this study) expressed in stably transformed ATE1 -KO MEF. The arginylation reporter protein, DD- 15-GFP, as described in Figure 4, was expressed and purified from atel I. yeast. On the right panel, Anti-RDD was used to probe the level of arginylation on the reporter protein. Anti-GFP was used to show the total amount of reporter protein (DD- 15-GFP) added in each sample. mAtel antibody was used to detect the level of mAtel.l-GFP (either WT or mutant) present in each sample. Actin was used as loading control for cell lysates. C) Graph showing the quantification of cell viability after treatment of different concentrations of STS for 12 hours, for WT and ATE1-KO cells stably expressing either GFP (as

transfection and expression control), GFP-fused recombinant mouse Atel. l, or

enzymatically impaired mutant mouse Atel. l. The viable cells were counted by cell counter and by using Trypan Blue to exclude dead cells. For each group, the data was normalized to a sample under non-stressing condition. Error bars represent SEM (n=3). D) Similar to C), except that a treatment of CdCl ₂ was used. Error bars represent SEM (n=3).

[0024] Figures 7A-7D. Knockout of ATE1 increases cell viability upon UV irradiation. A) Representative images of WT and atel I. yeast grown for 3 days after exposure to different doses of UV irradiation and recovery in the dark. B) Representative images showing WT or ATE1 -KO MEF after exposure to increasing doses of UV irradiation and recovery for 24 hours. C) Quantification of viable cells at 12 hours after UV treatment. Live cells were quantified with cell viability dye Calcein AM and the numbers were normalized to matching samples not irradiated (0 J/m ). Error bar represent SEM (n=3). D) Comparison of cell viabilities at 12 hours after UV treatment for WT or ATE1-KO cells stably expressing either GFP (as transfection and expression control), mAtel.l-GFP, or the catalytically-impaired mAtel.l-mut-GFP. The number of viable cells was directly counted by cell counter and by using trypan blue to exclude dead cells.

[0025] Figures 8A-8F. Knockout of ATE1 increases mutagenesis upon UV irradiation. A) Scheme showing the construction of a mutagenesis reporter plasmid. On the left is the vector map of the pHLU-M-stop plasmid, which contains three auxotrophic marker genes: HIS3, LEU2, URA3, and a mutated MET 15 gene with a stop-codon in the middle of its coding sequence. The scheme on the right shows a portion of the coding sequences and

corresponding amino acids in the original MET15 gene and the mutated Metl5-stop gene, where a TGG codon, coding for tryptophan (W), is converted to a TGA stop codon. B) The top panel shows a flow chart describing the procedure followed to create isogenic pairs of WT and atelA yeast and for testing emergence of Met-prototrophic mutant colonies on Met- minus plates starting with the same number of cells for UV irradiation. The bottom left panel has representative images showing the auxotrophic colonies emerged from 20 million yeast (in each spreading) without or with a low dose of UV exposure (50 J/m ). The graph on the bottom right is the quantification of the experiment on the left for all tested doses of UV irradiations. Error bar represent SEM (n=3, except for the control non-irradiated groups where n=6). C) Scheme showing the construction of a mammalian mutagenesis reporter, mCherryFP-STOP-IRES-GFP, which was modified from the pQC-XIG retroviral vector suitable for stable transfection. A STOP codon is inserted in the N-terminal region of the mCherryFP coding region so that this protein cannot be expressed as full-length, unless an acquired mutation reverts it to a sense codon (revertant). The scheme on the right shows a portion of the coding sequences and corresponding amino acids in the original mCherryFP gene and the mutated mCherryFP-STOP gene, where a TGG codon, coding for tryptophan (W), is converted to a TGA stop codon. D) Representative FACS charts showing the distribution of cell populations by their green and red fluorescence, for WT or ATE1 -KO MEF, in untreated condition or treated with low-dose UV irradiations that are not expected to lead to significant cell death (two pulses of 20 J/m irradiations over 48 hours, followed by 24 hour recovery). The windows marked "B" were the gate setting used to quantify and sort red- fluorescence-positive cells. E) Quantification of mutated cells showing a red fluorescent signal in FACS in untreated or UV-irradiated cells from 4 independent repeats (n=4). In untreated condition, both WT and ATE1 -KO cells has negligible numbers of revertants with no significant (ns, p>0.05) difference. After UV irradiations, while the WT cells have a moderate increase of revertants (-10 times), the increase in ATE1-KO is much larger (-100 times), resulting in a significant difference between the WT and KO cells. Error bar represents SEM. As mentioned before, the /?-value was calculated by Student's t-test. F) Representative fluorescent images of ATE1 -KO MEF stably expressing the reporter genes, either untreated, or treated with UV irradiation and enriched for red fluorescent cells by FACS for culturing of up to one week. In untreated cells, no red fluorescence presented in any examined cells. In UV-irradiated and sorted cells, the vast majority of the examined cells have prominent red fluorescence, in addition to the green fluorescence from the internal expression marker GFP on the reporter construct, indicating that they are true revertants. The white arrow in the image points to a false positive cell, which only has green fluorescence and not red fluorescence. Overall less than 5% of false positive was found in the examined cells.

[0026] Figure 9. The stress-induced increase of arginylation signal is dependent on the presence of Atel. As a negative control for Fig 3D, left side shows a similar procedure of using in-lysate arginylation assay on atel-Δ yeast exposed to increased duration of 1M NaCl stress. Right side shows a representative immunoblots probed with anti-RDD and anti-GFP, for the detection of arginylated form and the total level of the reporter protein DD- 15-GFP, respectively. The arrows indicated the expected position of the band of DD- 15-GFP on the blots. The protein molecular sizes were indicated with MagicMark XP protein marker (from Invitrogen), which can be visualized by chemifluorescence for positive labeling. As shown in the immunoblots, there was no signal, nor increase of signal, for arginylated DD- 15-GFP in the atel-Δ yeast with stress treatment. In addition, there was no sign of degradation of the substrate. These data suggest that the increase of arginylation signal observed in WT yeast shown in Fig. 3C is specifically mediated by Atel.

[0027] Figure 10. Validation of the reverting mutation by DNA sequencing. ATE1-KO cells stably with transfected mutation reporter mCherry-STOP were treated with UV- irradiation as shown in Fig.8 and the red-fluorescence positive cells were enriched by sorting and then grown for up to two weeks to reach sufficient cell numbers. Genomic DNA were extracted from those red-positive cells, as well as from untreated cells (as a control for sequencing). The DNA was amplified with two rounds of nested-PCR with two sets of primers that are specific for the region containing the mCherryFP sequence. The PCR products with anticipated size were then submitted for Sanger sequencing. On top of the figure, the original sequence of mCherryFP spanning the target site for mutation (underlined) was shown. In the middle is the sequencing result from untreated cells. Arrow points to the Trp (W)-STOP mutation as designed in the mCherry-STOP reporter. At the bottom is the sequencing result from the expanded culture of the pooled red-positive cells sorted from the UV-irradiated ATE1-KO cells carrying the mCherry-STOP vector. Arrows point to the sites of reverting mutation. It is worth noting that, based on the Sanger sequencing result, the detected inversion event is nearly exclusively A -> G, a purine-transition which reverts the STOP codon back to the original Trp codon. This could be due to the fact that a transition between purine, rather than a transversion between purine and pyramidine, is more common in genomic mutations [52]. Alternatively, it is possible that the Trp residue in this location is essential for the fluorescence of mCherryFP, so that only such a revertant can be detected and collected by FACS.

DETAILED DESCRIPTION

[0028] The disclosure provides methods for, e.g., detecting DNA mutagenesis. One such method includes introducing into a cell an expression vector comprising a nucleic acid sequence encoding a reporter protein fused to a substrate for arginyltransferase 1 (Atel), such that a reporter fusion protein comprising the reporter protein and Atel substrate is produced. The method further comprises measuring arginylation of the substrate and the reporter protein. The ratio of arginylated substrate to reporter protein is calculated. In an alternative embodiment, the method comprises (a) introducing into a cell a reporter plasmid comprising nucleic acids encoding a promoter, a first reporter protein, an internal ribosome entry site (IRES), and a second reporter protein. The first reporter protein and the second reporter protein are different proteins (i.e., the first and second reporter proteins are not both green fluorescent protein). Either the nucleic acid encoding the first reporter protein comprises a premature stop codon and mutation of the stop codon (e.g., to a sense codon) results in the expression of the first reporter protein, or the nucleic acid encoding the second reporter protein comprises a premature stop codon and mutation of the stop codon results in the expression of the second reporter protein. The method further comprises measuring the first reporter protein and the second reporter protein, and calculating the ratio of first reporter protein to second reporter protein. [0029] The dual reporter method described herein addresses inadequacies in previous methods. For example, when two reporter proteins are driven by one promoter and separated by an ribosome internal entry site (IRES), no difference in transcription is expected between the reporter proteins; the reporter proteins are located on the same expression vector (or integrated into the same genomic locus, if stable integration is used) and will be transcribed with the same transcription machinery. While a small difference in translational efficiency may occur between the two reporters, the difference does not result in a false signal because, e.g., one reporter protein serves as the mutation target while the other one is an internal control to validate the existence and transcriptional status of the reporter proteins. Also, because of this unique feature, the reporter assay described herein can be used to reliably and accurately compare mutation frequencies between different cell types and different organisms regardless of the difference in their transfection efficiency and transcriptional or translational activity, which cannot be reliably accomplished with pre-existing methods.

[0030] Various embodiments of the method of the disclosure involve detection of arginylation. Protein arginylation is a posttranslational addition of an extra arginine to an existing protein substrate, which is mediated solely by arginyltransferase 1 (Atel).

Arginylation changes primary sequence as well as surface charge of a protein and, therefore, potentially changes degradation half-life and functions of the target protein. In one embodiment, the disclosure provides a reporter fusion protein comprising an N-terminal sequence that is recognized by Atel as its substrate fused to a C-terminus reporter protein (e.g., GFP or other reporter protein that generates a signal that can be detected, preferably detected in an intact cell). The reporter fusion protein is compatible for in vitro and in vivo applications, which is an improvement compared to the peptide-based assay. The arginylated form of the recombinant fusion protein can be recognized with, e.g., an antibody that specifically recognizes the extra arginine within the substrate, while the total amount of the recombinant fusion protein can be measured by another antibody that specifically recognizes the reporter protein subunit of the fusion protein. The ratio of these two signals is then calculated to reflect the actual arginylation extent of the reporter substrate. The use of two antibodies in various embodiments to confirm the arginylation level of the reporter protein increases the specificities and accuracy of this assay. This method is applicable for, e.g., Western Blot format, which allow the user to specifically detect the arginylation signal of the reporter without confusion with other proteins. It can also be used in an ELISA format, immunofluorescence, or any other antibody-based methods. In addition, because two antibodies are used in this embodiment, the method can be used for applications that require close proximity of two or more antibodies to generate a signal for readout, such as, for example, the Alpha Technology/ AlphaScreen.

Exemplary Embodiments and Examples

[0031] The present disclosure reveals that Atel and Ate 1 -mediated arginylation are up- regulated as part of the general stress response to induce cell growth-arrest and cell death, which results in the prevention of DNA mutagenesis under stress. Furthermore the disclosure clarifies the nature of the involvements of Atel and its arginylation activity in stress response, potentially providing a novel explanation for the in vivo functions of Atel and arginylation in a host of human diseases. Many of these stressing conditions are known to have causative effects for human diseases such as cancer, cardiovascular disease, aging, developmental abnormalities, injury and inflammation. Biochemical and genetic evidence have shown a fluctuation of arginylation activity or disruption of ATE1 gene being correlated with some of these diseases [2, 3, 9-18, 41-44]. Tumor microenvironments are often highly stressing due to poor formation of blood vessels or high activity of mitogenic factors. Data herein indicates that down-regulation of Atel /arginylation would assist cancer cells to survive the stress, and to accumulate mutations, thereby increasing tumor growth and metastatic risks.

[0032] The below described embodiments illustrate representative examples of methods of the disclosure. From the description of these embodiments, other aspects of the invention can be made and/or practiced based on the description provided below. The methods involve use of immunological and molecular biological techniques described in methodology treatises such as Current Protocols in Immunology, Coligan et al., ed., John Wiley & Sons, New York. Techniques of molecular biology are described in detail in treatises such as Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Sambrook et al., ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001 ; and Current Protocols in Molecular Biology, Ausubel et al., ed., Greene Publishing and Wiley-Interscience, New York. General methods of medical treatment are described in McPhee and Papadakis, Current Medical Diagnosis and Treatment 2010, 49th Edition, McGraw-Hill Medical, 2010; and Fauci et al., Harrison's Principles of Internal Medicine, 17th Edition, McGraw-Hill Professional, 2008.

Production of custom antibody against ROD peptide (anti-RDD) [0033] Rabbit polyclonal anti-RDD was ordered from Genscript INC. by custom antibody production protocol optimized for recognition of post-translational modifications. In brief, a custom synthesized peptide with the sequence of RDDIAALVVDC (SEQ ID NO: 1) (from Genscript) was conjugated through the C-terminal cysteine to a carrier protein, keyhole limpet hemocyanin (KLH), to increase the presentation of the N-terminus of the peptide. The conjugated protein was used as the immunogen for repeated immunization in rabbits. The harvested antisera was cross-absorbed by another synthetic peptide with the sequence of DDIAALVVDC (SEQ ID NO:2) (from Genscript). The resulted antisera (anti-RDD) was confirmed by immunoblots to have minimal cross -reactivity to the peptide DDIAALVVDC (SEQ ID NO:2), or the N-terminally acetylated peptide Ac-DDIAALVVDC (SEQ ID NO:2) (a gift from Dr. Anna Kashina in the University of Pennsylvania). shRNA-mediated knockdown ofAtel

[0034] p.LKO lentiviral vectors containing shRNA targeting GFP, mouse Atel, and human Atel were obtained from the Mission shRNA catalog (Sigma; clone NM_007041.1-520slcl for human; clone NM_013799.2-1507slcl for mouse). These vectors were packaged using VSV.g and Δ8.2 lentivirus packaging vectors by co-transfecting HEK293T cells with the aid of transfection kit Lipofectamin 2000 (Life Technologies). The viral supernatant was collected at 24, 48, and 72 hours, and filtered through a 0.45μΜ Syringe filter (Olympus, #25-246). The supernatant was then used to transduce MEF and HFF cells, aided by 10μg/mL polybrene. After transduction, 5μg/mL puromycin was used to select transduced fibroblast cells for 5-10 days. Once the selected line was stabilized, a Western blot was run on a cell lysate to examine changes in Atel levels.

Stress tests and radiation treatment of mammalian cells

[0035] To evaluate the survival of MEFs and HFFs to stress and radiation, 10 ⁴ cells were plated in each well of a 94 well black-wall plate, with 4 replicates per dose. The cells were allowed to attach for 4-6 hours. For the Η ₂ 0 ₂ treatment, H ₂ 0 ₂ (Sigma, Cat# 323381) was added to the cells for 12 hours or indicated durations. The cells were then washed with PBS, and cell viability was assessed via Calcein AM staining. For the UV irradiation assay, the culture medium was removed immediately before the irradiation and immediately added back afterward. The cells were then allowed to recover for 24 hours at 37°C, in a C0 ₂ incubator before examination or the next treatment. The cell viability was either assessed via staining of live-cell dye Calcein AM or by direct counting in an automated cell counter TC20 (Biorad) with the aid of cell viability dye Trypan Blue.

In Vitro Arginylation Assay

[0036] For in vitro arginylation with yeast extract, exponential growth-phase cells of WT BY4741 -strain yeast (either transfected or not) were used. Equal number of cells treated and not treated with test conditions were lysed in arginylation reaction buffer (50mM Hepes, pH 7.5, 25mM KC1, 15mM MgC12, 2.5mM ATP, Arginine 0.2mM, PMSF 0.1M, and yeast protease inhibitor cocktail (final dilution lOOx) (Sigma- Aldrich)[37]. The atelA yeast expressing DD-P15-GFP were also lysed in arginylation buffer. The WT cell lysate was mixed with equal volume of lysate of atelA cells expressing the DD-P15-GFP to start the arginylation reaction. The reaction was carried out for 10 minutes at 37°C. The reaction was stopped by the addition of 1/3 volume of 4x SDS sample buffer and boiling. For in vitro arginylation with extract of mammalian cells (transfected or not), exponential growth-phase cells of MEF were harvested from culture plate, washed by dPBS, and then weighed on scale. The cell pellets were lysed with 2x volume of a modified reaction buffer (50mM TRIS/HCl, 32mM Na ₃ P0 ₄ , pH 7.4, 5mM MgCl ₂ , ImM EDTA, 2.5mM ATP, 0.2mM Arginine, with 0.2% NP-40). As an arginylation reporter, recombinant protein DD- 15-GFP was expressed in atelA yeast and then purified by GFP-TRAPS nanobody conjugated to magnetic beads (Bulldog Bio) and was shown to be more than 95% pure by Coomassie-blue staining in SDS- PAGE. This protein was added to the cell lysate as the substrate for arginylation. The reaction is allowed to proceed in room temperature for 45 minutes, and terminated by addition of equal volume of 4x SDS sample buffer and boiling for 10 minutes.

Mutagenesis detection with dual-color reporter in mammalian cells

[0037] Methods of using the reporter plasmid as described in this patent application may include the following. MEF (WT or ATE1-KO) were stably transfected with the dual-color mutation reporter mCherryFP-STOP-IRES-GFP. Equal numbers of each type of cells were inoculated on 150mm-diameter cell culture dish for 4-6 hours to allow attachment. The final cell density did not exceed 25% confluency. Immediately before UV irradiations, the culture medium is removed. After UV treatment, the original culture medium was added back to the dish and the cells were allowed to recover for 24 hours in C0 ₂ incubator at 37°C before another round of UV irradiation. After two rounds of UV irradiations, the cells were allowed to recover for 24 hours, before FACS analysis was performed on viable cells that remained attached to the dish.

Removal or down-regulation ofAtel disrupts stress response and reduces cellular sensitivity to a variety of stressing conditions

[0038] Atel is coded by a single gene in yeast and mammals [4, 5]. When deleted the evolutionary conserved ATE1 gene in the budding yeast, S. cerevisiae (strain BY4741, unless otherwise indicated), no obvious effect on growth in non-stressing conditions in nutrient-rich medium (Fig. 1A, B). Upon exposure to stress, including H ₂ 0 ₂ -induced oxidative stress, heavy metals, high salt, or high temperature, wild-type (WT) yeast grew at a significantly lower rate compared to non-stressing conditions, which is an expected outcome of normal stress response (Fig. 1A and IB). However, the growth of atel A yeast was less affected by these stressors (Fig.lA and IB), suggesting a disruption of stress response [36]. The deletion of ATE1 gene in a different yeast strain, W303, similarly increased cellular resistance to CdCl ₂ (Fig. 1C).

[0039] To test whether Atel has a similar role in mammals, WT was compared with ATE1- KO mouse embryonic fibroblasts (MEF) and the deletion of ATE1 gene was found to be increased cellular resistance to stressors such as cellular oxidant H ₂ 0 ₂ , heavy metal CdCl ₂ , and microbial alkaloid toxin staurosporine (STS) (Fig. ID). In addition, when ATE1 expression was attenuated by shRNA-mediated knockdown in MEF and human foreskin fibroblasts (HFF) (Fig. IF), resistance to the oxidant H ₂ 0 ₂ was increased (Fig. IE and IF).

[0040] Common effects of stress response include cell death and growth-arrest. To test whether the deletion of ATE1 affects cell death in yeast, cellular viabilities by colony- formation unit (CFU) in yeast cultures in the presence of lethal doses of H ₂ 0 ₂ were examined. Atel Δ yeast cultures were found to have higher percentages of viable cells compared to the WT (Fig. 2A). Furthermore, by using the TUNEL assay to probe apoptosis, a programmed cell death event, the deletion of ATE1 greatly attenuated H ₂ 0 ₂ -induced apoptosis (Fig. 2B), which contradicts the prevailing hypothesis for the anti-apoptotic roles for Atel and arginylation [23, 28, 29].

[0041] To examine whether Atel also impacts growth-arrest, yeast cultures were exposed to a moderate concentration (150μΜ) of CdCl ₂ . This treatment led to a much lower growth rate in the WT yeast culture compared to atel Δ (Fig.2C). However, both cultures have similar viabilities as assessed by the CFU assay (Fig.2D). In fact, in the late sampling time- point (24 hours), the WT yeast even appeared to have slightly higher viability compared to atelA, probably due to nutrient constraints in the atelA yeast culture because it reached a saturation density at that time point. Therefore, the faster growth rates of atelA yeast shown in this test was likely caused by a lack of growth-arrest.

[0042] To further test whether Atel affects both growth-arrest and cell death for the same stressor, the WT and atel A yeast were challenged with high-temperature conditions. At 40°C, a heat-stress temperature for yeast, the atel A yeast grew significantly faster than the WT. However, when these yeast cultures were transferred from 40°C to room temperature (RT), a non-stressing temperature for recovery, similar numbers of colonies eventually formed in both the atel A and WT yeast. This suggests that both yeast strains were equally viable, and their difference in growth at 40°C was mainly due to a difference in growth-arrest (Fig.2E). However, when yeast were incubated at 42°C and then transferred to room temperature, atel A yeast were able to form significantly more colonies than the WT yeast, indicating that a deletion of ATE1 in this condition yielded a higher cell survival rate (Fig.2F). Therefore, the lack of Atel may lead to the bypass of growth-arrest and/or the suppression of cell death from the same stressor, dependent on the intensity of stressor.

[0043] To test whether mammalian Atel (mAtel) mediates apoptosis, WT and ATE1-KO MEF were challenged with apoptosis-inducing reagent STS of relatively high doses (200 to ΙΟΟΟηΜ) for a short duration (5 hours). By employing the Annexin-V staining assay to detect early apoptotic signals, the knockout of ATE1 gene in MEF significantly reduced apoptotic rates in the presence of STS (Fig. 3A). Similar results were observed with a different stressor, CdCl ₂ (Fig.3A). Next, to test whether mammalian Atel regulates growth-arrest in stressing conditions, the WT and ATE1 -KO MEF were challenged with either H ₂ 0 ₂ or CdCl ₂ , with lower doses and longer durations (12 hours). By using a thymidine analogue, 5-Ethynyl-2'- deoxyuridine (EdU) to measure cell proliferation activity, no significant difference was found between WT and ATE1-KO MEF in non-stressing conditions. However, in the presence of stressors, the percentage of EdU-positive population in WT cells is significantly lower than in ATE1-KO MEF, suggesting that the absence oiATEl prevents growth suppression of mammalian cells under stress (Fig.3B).

Atel and arginylation are up-regulated during stress and are responsible for cell death [0044] In yeast and mammals, only one arginyltransferase (ATE1) was identified [4, 5]. To confirm that the deletion of this gene can abolish cellular arginylation activity, a reporter substrate termed DD-P15-GFP was designed (Fig. 4A). When this reporter protein was expressed in yeast cells, it was observed that its arginylated form existed only in WT yeast, but not in atelA (Fig. 4B). A similar observation was made in mammalian cells MEF (Fig. 4E). As a minor note, the steady state level of the reporter protein, as probed by anti-GFP, was obviously lower in WT yeast than in atelA, likely due to an arginylation-mediated degradation in yeast (Fig. 4B).

[0045] In most past studies, arginylation was demonstrated by radioactively labeled arginine, which can be incorporated into protein by arginylation or translation. To separate these two effects, translation was inhibited by either inhibitors or the depletion of ribosome [9-14]. However most translation inhibitors are unfortunately leaky, and removal of ribosome can collaterally deplete Atel due to their known affinity [37]. To remove these ambiguities, DD- 15-GFP was used for an "in-lysate" reaction to examine arginylation activity in cell extracts (Fig.4C left panel). By using this assay, an increase of arginylation signal in WT was observed when yeast were exposed to NaCl as an osmotic stressor, in proportion to stress durations (Fig. 4C right panel). Also, the level of the substrate is reduced proportionally, likely due to the arginylation-mediated degradation (Fig. 4C right panel, middle strip). As a control, extracts from atel A yeast exposed to the same treatment did not show an increase (or any signal) of the arginylated reporter or a reduction of the substrates (Fig. 9), suggesting that the stress-induced increase of arginylation depends on the presence of Atel. Consistent with the observation in yeast, a dose-dependent increase of arginylation activity was found in MEF treated with H ₂ O ₂ (Fig. 4E). These data indicates that the global arginylation activity in the cell is up-regulated as part of the natural stress-response process, which is consistent with previous observations [9-14].

[0046] To test whether Atel protein is also up-regulated in stress response, a commercially available yeast strain carrying an "z ^' n locus" 3 '-end fusion of GFP with the endogenous ATE1 gene (termed "endo: Atel-GFP") under the control of the endogenous ATE1 promoter, was used. The protein level of "endo: Atel-GFP" was proportionally increased with the dose of each stressors such as H ₂ O ₂ or high salt, (Fig. 4D). Similarly, probing of the steady-state level of endogenous Atel in MEF, resulted in a dose-dependent increase of total Atel protein in the presence of H ₂ O ₂ , (Fig. 4F). These data suggest that Atel protein level is up-regulated during stress in yeast and mammalian cells.

[0047] Since cell death is a common outcome of extended stress response [36], whether the up-regulation of Atel/arginylation could be a direct inducer of cell death was investigated. To specifically test the effects of Atel on cell death, a galactose-inducible recombinant Atel (termed "pGALl: Atel ") plasmid was used in yeast. To avoid interference of the endogenous Atel, atel A yeast were transformed with this plasmid (or an empty vector as control). The growth of yeast carrying the inducible Atel was strongly repressed on galactose-containing plate, where the expression of the recombinant protein was turned on (Fig. 5A). A similar effect was observed with the induced-expression of a GFP-fused Atel (pGAL: ATE1-GFP) (Fig. 5E). To verify whether the increased abundance of Atel can induce cell death, yeast cells were induced in galactose-containing liquid medium for increasing durations. The cell viability was then measured by a CFU assay in glucose-containing agar plates (where the galactose-induction is terminated). It was observed that that the viability of cells carrying the pGAL: ATE1-GFP was dramatically decreased along the time line of galactose induction, suggesting that the up-regulation of Atel is indeed capable of inducing cell death in yeast (Fig. 5B).

[0048] To further correlate Atel effects on stress response with its arginylation activity, two critical cysteine (C) residues (20 and 23) were replaced with serine (S) on Atel. These mutations were reported to minimize the enzymatic activity of Atel without affecting its solubility, based on in vitro studies [38]. Consistently, the expression level of the C20,23S mutant Atel was similar to the WT version of Atel, suggesting that these mutations did not affect the overall synthesis and turnover of this protein (Fig. 5C). To verify whether such mutations indeed reduce Atel activity [37], the in-lysate arginylation assay was utilized with DD- 15-GFP reporter and it was found that the activity of the mutation is less than 25% of the WT enzyme (Fig. 5D). When the cells over-expressing the mutant Atel and the WT Atel were compared, the C20,23S mutation was found to significantly reverse the repressing effects of Atel over-expression in cells (Fig. 5E). Therefore, the effects of yeast Atel on stress response is largely (if not completely) dependent on its arginylation activity.

[0049] To test whether the mammalian Atel has similar effects, splicing variant 1 of mouse Atel was used (referred as mAtel.l), the most ubiquitous isoform that exists in all tissues of animals [39], as the representative. The C-terminal GFP fused recombinant protein was reintroduced into ATE1 -KO MEF by a stable low-expression system that generates recombinant proteins at a level comparable to the endogenous level of WT Atel (Fig.

6A)[40]. The expression of mAtel. l was able to reinstall cellular sensitivity to stressors, such as STS and CdCl ₂ (Fig. 6C and 6D), to levels close to WT cells, indicating that mammalian Atel is indeed mediating cell death in stress response. Mammalian Atel also carries two cysteine residues (C23, C26 in mAtel) corresponding to the C20, C23 residues in yeast Atel. Consistent with our observations in yeast Atel, when these two cysteine residues were changed to serine in mouse Atel, the resulted mutant (mAtel.1-mut) had compromised arginylation activity (Fig. 6B), and had significantly lower capacity to reinstall cellular sensitivity to STS or CdCl ₂ than the original mAtel.l (Fig. 6C and 6D). These data indicate that the arginylation activity of mammalian Atel is also required for its action in mediating stress response.

Atel is essential for the suppression of mutagenesis during DNA-damaging stress

[0050] Growth-arrest and cell death during stress could be interpreted as a mechanism to prevent incorporation of damaged genetic material or transmission of mutation to the subsequent generations. This would raise the question whether Atel, as a posttranslational modification enzyme, has the capacity to affect the outcome of mutagenesis.

[0051] To test whether Atel affects cellular response to DNA-damaging stress, cells were subjected to ultra-violet (UV) light, a common and natural mutagenic source that generates DNA damage as well as various stressing signals in cells. The ATE1 -deletion in yeast or MEF was found to significantly increased their resistance to UV light (254 nm UV-C) compared to WT (Fig. 7A, 7B, and 7C). In addition, the sensitivity to UV in ATE1-KO MEF can be sufficiently reinstalled by the reintroduction of recombinant Atel (mAtel. l). This installation effect was nearly abolished when the catalytically impaired mutations (C23-26S) was introduced in Atel (Fig. 7D).

[0052] To test whether Atel is required to prevent DNA mutations in yeast upon UV irradiation, a mutation reporter Met-STOP was designed, in which a Tryptophan (TRP/W) residue in Metl5 gene is replaced with an interrupting STOP codon. The product of this engineered gene is not expected to rescue the methionine (Met)-auxotrophy phenotype of

BY4741-yeast, unless an acquired mutation reverts the interrupting STOP codon to a sense codon. (Fig. 8A). During non-stressing conditions, it was observed that WT and

atel A BY4741 -yeast carrying this reporter plasmid similarly generated negligible numbers of colonies on culture plates in the absence of methionine. However, after exposure to UV irradiations, atelA yeast produced significantly more colonies acquiring Met-prototrophic phenotype compared to the WT, suggesting that the deletion of Atel lead to higher mutation frequencies (Fig. 8B). Since significant differences between WT and atel A yeast were observed in both low and high doses of UV irradiation, it is likely that both the effects of Atel in growth-arrest and cell death contribute to the mutation suppression.

[0053] To validate these observations in mammalian cells, a dual-color mutation reporter system mCherryFP-STOP-IRES-GFP was created, in which an interrupting STOP codon replaces a Trp residue in mCherryFP gene, which is followed by an internal ribosome entry site (IRES) and a green fluorescent protein eGFP (as the internal control for transcription and expression). As such this reporter is expected to generate a green fluorescence but not a red- fluorescent signal unless a new mutation reverts the interrupting STOP codon in mCherryFP to a sense codon (Fig. 8C). To measure the emergence of mutants, the WT or ATEl-KO MEF stably transfected with the reporter, either treated with UV irradiation or not, were analyzed in fluorescence flow cytometry (FACS). In non-stressing conditions, both the WT and ATEl- KO cells had negligible numbers of red fluorescent cells. However, when exposed to a low dose of UV irradiation not expected to lead to significant cell death, a significantly higher ratio of red fluorescent cells was detected in the ATEl-KO cells compared to the WT cells (Fig. 8D and 8E). To further validate that the red fluorescence detected by FACS was genuine, the positive cells were sorted and subsequently examined by microscopy. It was found that the majority of collected cells exhibited both red and green fluorescence, with less than 5% being false positives having only green fluorescence (Fig. 8F). Finally, to confirm the mutation in the reporter gene, sequencing was performed on the corresponding region with genomic DNA prepared from these red-positive cells and identified a reverting mutation in the anticipated location, which was absent in the untreated cells (Fig. 10).

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