Bad Smells and Broken DNA: A Tale of Sulfur-Nucleic Acid Cooperation

Hydrogen sulfide (H2S) is a gasotransmitter that exerts numerous physiologic and pathophysiologic effects. Recently, a role for H2S in DNA repair has been identified, where H2S modulates cell cycle checkpoint responses, the DNA damage response (DDR), and mitochondrial and nuclear genomic stability. In addition, several DNA repair proteins modulate cellular H2S concentrations and cellular sulfur metabolism and, in turn, are regulated by cellular H2S concentrations. Many DDR proteins are now pharmacologically inhibited in targeted cancer therapies. As H2S and the enzymes that synthesize it are increased in many human malignancies, it is likely that H2S synthesis inhibition by these therapies is an underappreciated aspect of these cancer treatments. Moreover, both H2S and DDR protein activities in cancer and cardiovascular diseases are becoming increasingly apparent, implicating a DDR–H2S signaling axis in these pathophysiologic processes. Taken together, H2S and DNA repair likely play a central and presently poorly understood role in both normal cellular function and a wide array of human pathophysiologic processes. Here, we review the role of H2S in DNA repair.


Introduction
The maintenance of genomic stability is essential for life, and cells have evolved complex and intricate molecular machinery to ensure DNA stability and accurate DNA replication [1][2][3][4]. Eukaryotic cells carry two separate genomes with different evolutionary origins [4,5]. The nuclear genome is diploid, linear, and in humans contains roughly 3.3 billion base pairs encoding over 20,000 genes [4,5]. Conversely, the mitochondrial genome is circular, contains 37 genes in 16,569 base pairs, and occurs in multiple copies at 100-1000/cell [4,5]. The two genomes extensively interact, with the nuclear genome encoding roughly 1500 mitochondrial proteins, including those involved in mitochondrial DNA repair, while mitochondrial genomic damage can initiate apoptotic cell death via cytochrome c release and can also activate the innate immune response [4][5][6][7]. Hydrogen sulfide (H 2 S) is a gasotransmitter that, along with nitric oxide and carbon monoxide, functions in a vast number of different physiologic and pathophysiologic processes [8,9]. Specifically, H 2 S has many physiologic regulatory roles, including in the renal, cardiovascular, central nervous, and digestive systems, and is also dysregulated in many different pathologic processes including cancer, cardiovascular diseases, and neurodegeneration [10][11][12][13][14][15][16][17]. Recently, H 2 S has been found to regulate mitochondrial and nuclear DNA stability and repair [11][12][13].
Here, we review this new area of inquiry and discuss its possible implications for cancer chemotherapy and cardiovascular diseases.
The enzymatic synthesis of H 2 S in mammals requires methionine and cysteine, with methionine being derived solely from the diet, as it cannot be synthesized in mammalian tissues [19,34]. These amino acids are converted into homocysteine by the transsulfuration pathway [19,34]. CBS synthesizes H 2 S by catalyzing homocysteine and L-cysteine to form cystathionine and H 2 S [19,35]. CBS generates H 2 S through a β-replacement reaction also producing serine. In the presence of homocysteine H 2 S synthesis increases 23-fold compared to the reaction with L-cysteine alone [19,36]. CBS also catalyzes the condensation of L-serine and homocysteine, forming cystathionine and H 2 O, a significant step in L-cysteine biosynthesis [37]. CSE catalyzes homocysteine, generating H 2 S, α-ketobutyrate, and NH 3 [9,19,38]. Interestingly, at physiologic L-cysteine and homocysteine concentrations, roughly 70% of the H 2 S synthesized for the CSE-mediated α,β-elimination of L-cysteine, while the α,γ-elimination of homocysteine contributes approximately 29% of the total H 2 S content; however, roughly 90% of the H 2 S is derived from the α,γ-elimination of homocysteine when its levels are increased to those seen in hyperhomocysteinemia [38]. 3-MST H 2 S synthesis requires cysteine aminotransferase to convert L-cysteine into 3mercaptopyruvate, which is then catalyzed by 3-MST into H 2 S and pyruvate [39]. Dcysteine can be converted to 3-mercaptopyruvate by the peroxisome-located D-amino acid oxidase, which, once imported into the mitochondrion, is converted into H 2 S by 3-MST. As D-amino acid oxidase expression is limited to the brain and kidneys, this H 2 S synthesis pathway is likely limited to those organs [40].
Based on its chemistry, the molecular mechanisms of H 2 S reactivity have been placed into three categories: (1) chemical interfacing/scavenging with reactive oxygen and nitrogen species, (2) chemical modification of protein cysteines to persulfides, and (3) binding to and/or redox reactions with metal centers [9]. Intracellular H 2 S exists as free H 2 S, acidlabile sulfide, and bound sulfane sulfur [19]. The acid-labile sulfide faction consists of sulfur present in iron-sulfur clusters contained within iron-sulfur proteins (non-heme), which are ubiquitous and include ferredoxins, rubredoxins, aconitase, and succinate dehydrogenase [19]. The sulfane sulfur fraction consists of sulfur atoms bound only to other sulfur atoms, these include thiosulfate S 2 O 3 2-, persulfides, R-S-SH, thiosulfonates R-S(O)-S-R', polysulfides R-S n -R, polythionates S n O 6 2-, and elemental sulfur S 0 [19]. Free cellular H 2 S represents less than 1% of the potentially available sulfide, indicating that the endogenous sulfide pool likely has significant buffering capacity [9].

Non-Enzymatic H 2 S Synthesis
H 2 S is also synthesized through non-enzymatic mechanisms and occurs by an iron and vitamin B 6 -mediated catalysis of L-or D-cysteine producing pyruvate, NH 3 , and H 2 S [41]. This H 2 S synthesizing pathway likely plays a role in maintaining basal H 2 S levels and may be an important H 2 S source in iron overload and hemorrhagic disorders [41]. Lastly, the mammalian microbiome regulates systemic H 2 S bioavailability and metabolism. Germ-free mice show significantly lower plasma and gastrointestinal H 2 S and 50-80% lower plasma, adipose, and lung tissue bound sulfane sulfur compared to conventionally housed mice. Interestingly, CSE activity was reduced in many organs of the germ-free mice, while tissue cysteine levels were elevated [42].

H 2 S Catabolism
High cellular H 2 S concentrations can be toxic, and excess H 2 S is predominately removed by the stringently regulated mitochondrial sulfur oxidation pathway [43]. H 2 S catabolism is initiated by the mitochondrial matrix flavoprotein sulfide quinone oxidoreductase (SQR), which oxidizes H 2 S to form an SQR-persulfide intermediate. The persulfide is then transferred to glutathione to form glutathione persulfide, which is further oxidized by the ethylmalonic encephalopathy 1 or thiosulfate sulfurtransferase proteins to form sulfite or thiosulfate, respectively [43][44][45]. The sulfite and thiosulfate are ultimately excreted in the urine [46]. The electrons released by SQR enter complex III of the electron transport chain and are used to generate ATP, making H 2 S an inorganic compound capable driving mitochondrial ATP synthesis [47]. Outside of this review, however, polysulfides can be synthesized by all three H 2 S-sythesizing enzymes and increasingly have been found to play important functions in many physiologic and pathophysiologic processes [29,48,49].

Life's Origin and H 2 S
Biochemical, fossil, and molecular clock dating methods indicate that life first appeared 3.7 to 4.2 billion years ago in a reducing, ferruginous, and euxinic environment probably at hydrothermal vents rich in NH 3 , N 2 , CO 2 , CO, CH 4 , H 2 , H 2 S, and dissolved metals, especially Fe 2+ and Mn 2+ [50][51][52][53][54][55]. Due to the fact of its ubiquity on the early Earth and versatile chemistry, H 2 S likely played an essential role in prebiotic chemistry and the emergence of life [52,54,55]. Support for this comes from analyses demonstrating that reactive oxygen and reactive sulfur species detoxifying mechanisms have been present since the origin of life, some four billion years ago, and have continued to the present in the biochemistry of the Archaea, Bacteria, and Eukarya [50][51][52][53][54][55][56]. Since genomic stability maintenance is an ancient and absolute requirement for life, and H 2 S biochemistry is similarly ancient and ubiquitous, it is very likely that H 2 S functioned in the earliest biochemical pathways including in those regulating genomic stability [1][2][3][11][12][13]17,[52][53][54][55][56][57][58].

H 2 S and the DNA Damage Response
The DNA damage response (DDR) comprises a complex network of cellular pathways that cooperatively detect DNA damage, signals its presence, and promotes DNA repair, maintaining genomic stability [1][2][3]. Phylogenomic analyses indicate that many elements of the Eukaryotic DDR are ancient, appearing in the first Metazoa and subsequently undergoing evolutionary diversification [59].
Extensive evidence indicates that H 2 S affects cell DNA stability, impinging on the DDR and cell viability. For example, when the nuclei from Chinese hamster ovary cells were treated 2 h with 1 µM Na 2 S, they exhibited significant DNA damage that was attenuated by treatment with the antioxidant butyl-hydroxyanisole [60]. Treatment of two glioblastoma cell lines with a high amount of Na 2 S (476 µM) for 4 h, increased DNA damage, oxidative stress levels, and increased γ-H2AX foci formation [61]. Additionally, treatment of human intestinal epithelial cells with very high (1-2 mM) Na 2 S induced DNA breaks as measured by the comet assay [62]. Similarly, human lung fibroblasts treated 12 h with 10 µM NaHS showed micronuclei formation, increased p21, p53, Bax, cytochrome c, Ku-70 and Ku-80 expression, and a G 1 checkpoint response [63]. These studies implicate H 2 S in the DDR as; (1) broken DNA activates the DDR, (2) γ-H2AX foci formation requires the activities of the DDR proteins ATM, ATR, and DNA-PK, (3) the Ku70/Ku80 heterodimer associates with DNA-PK to promote DNA repair, and (4) the ATM kinase is required for the oxidative stress-induced G 1 checkpoint response and rapid p53 induction [1][2][3]64].
Other studies have shown that H 2 S can increase/preserve DNA stability. For example, in a murine model, a unilateral nephrectomy with contralateral ureteral obstruction suppressed H 2 S kidney levels and caused more DNA damage in CSE deficient mice compared to wild-type mice, indicating that CSE expression plays a role in maintaining DNA stability upon ischemia/reperfusion injury [65]. Additionally, daily injection of the H 2 S donor diallyl sulfide intraperitoneally into female rats at 50 mg/kg induced p53, Gadd45a, PCNA, and DNA polymerase δ in their breast tissue, suggesting that H 2 S enhances breast tissue DNA repair capacity [66]. Interestingly, exogenous H 2 S also affects the mitochondrial genome. For example, CSE deficient murine smooth muscle and aortic tissue showed reduced mitochondrial DNA copy numbers, mitochondrial content, mitochondrial-specific mRNAs (MT-CO1, CytB, and Atp 6), and mitochondrial transcription factor A (TFAM) mRNA and protein expression, and it elevated DNA methyltransferase 3A (Dnmt3a) expression, accompanied by increased global DNA methylation with increased TFAM promoter methylation [67]. Treatment with 30 and 60 µM NaSH for 48 h reversed these effects, with increased mitochondrial marker expression (mitochondrial DNA copy numbers, mRNAs, and mitochondrial content) and decreased Dnmt3a and TFAM promoter methylation, increasing TFAM expression [67]. This study indicates that CSE-derived H 2 S plays an important role in the maintenance of mitochondrial function and genomic stability. Lastly, 30-100 nM concentrations of AP39, a mitochondria-targeted H 2 S donor, increased endothelial cell H 2 S levels and stimulated mitochondrial electron transport and bioenergetic functions. Treatment of the endothelial cells with oxidative stress increased reactive oxygen species (ROS), reduced cell viability, suppressed cellular bioenergetics, and increased mitochondrial DNA damage, events reversed by 100 nM AP39 treatment [68]. Taken together, these studies indicate that under different conditions H 2 S can increase DNA damage or suppress it and also impinges on the DDR. A caveat to keep in mind is that many experimental procedures in these studies used high concentrations of H 2 S donors that are likely non-physiologic [61,62]. Additionally, many studies of H 2 S biology use H 2 S synthesis inhibitors that have low specificity, complicating experimental result interpretation (reviewed in [69]).

H 2 S and Mitochondrial DNA Repair
The mitochondria are the major cellular site for ROS generation, and the mitochondrial genome is subject to significant DNA, protein, and lipid oxidative damage [70]. Mitochondrial DNA repair is distinct from and, in general, less complex than the nuclear DNA repair systems. For example, base-excision repair (BER) predominates, while nucleotide excision repair (NER) is absent [5,71]. Moreover, mitochondrial genomes with double-stranded DNA (dsDNA) breaks are usually rapidly degraded, leading to a drop in genome copy number, which are replaced through non-cleaved genome replication, often leading to a shift in heteroplasmy [5]. A role for H 2 S in mitochondrial function is well established with, for example, the mitochondrial H 2 S donor AP39 promoting mitochondrial bioenergetics and genomic stability and in the face of exogenous oxidants [68]. Additionally, in ovarian cancer cell lines, CBS expression maintains mitofusin-2 expression, with CBS knockdown lowering mitofusin-2 expression, causing mitochondrial fragmentation with a fused spherical morphology and increased unbranched mitochondria [72]. Mitofusin-2 exerts anti-apoptotic effects, and its ablation is lethal in mice [73]. Interestingly, its expression is lower in obesity, diabetes, and in animal models prone to atherosclerosis, and is increased by weight loss and exercise [73].
The apyrimidinic/apurinic endonuclease 1 (APE1), exonuclease G (EXOG), DNA Ligase III (LIG3), and DNA polymerase gamma (Pol γ) play central roles in mitochondrial BER [4,11,[74][75][76]. Loss of these proteins has severe often lethal effects. For example, EXOG depletion induces persistent single-stranded DNA breaks leading to apoptosis, while APE1 ablation is embryonic lethal, and its removal by Cre expression causes apoptotic cell death within 24 h [74,75]. In the A549 lung adenocarcinoma cell line, siRNA knockdown of CBS, CSE, or 3-MST or treatment with the CSE-specific inhibitor D, L-propargylglycine (PAG) combined with exogenous oxidative stress significantly increased mitochondrial DNA damage [11]. Interestingly, the interactions of EXOG with APE1, LIG3, and POL were all attenuated with CBS, CSE, or 3-MST knockdown or pharmacologic CBS inhibition by aminooxyacetic acid (AOAA) [11]. The interactions of EXOG with APE1 or LIG3 following AOAA treatment were restored and mitochondrial DNA damage was reduced with AP39 co-treatment, demonstrating that mitochondrial H 2 S restored these interactions and increased mitochondrial genomic stability [11]. Mass spectrometric analysis revealed that EXOG Cys 76 was sulfhydrated, with the H 2 S donor NaHS increasing EXOC and APE1 interactions. Mutation of EXOG Cys 76 to alanine lowered its interactions with APE1 and made the interaction insensitive to NaHS treatment [11]. Thus, this elegant study demonstrated that mitochondrial H 2 S plays a central role in mitochondrial genomic stability and DNA repair.

ATR
Nuclear DNA repair and the DDR involve at least five major pathways comprising BER, NER, mismatch excision repair, homologous recombination, and non-homologous end joining [1][2][3]. The ataxia-telangiectasia mutated, and RAD3-related serine/threonine protein kinase (ATR) plays a central role in the DDR, where it stabilizes single-stranded DNA (ssDNA) at stalled replication forks, lowers replication stress, initiates cell cycle checkpoints, and promotes faithful anaphase chromosomal segregation [1][2][3]12]. Interestingly, increased ATR/phospho-ATR expression is a poor prognostic factor in breast, bladder, and ovarian cancers [77][78][79]. Analysis of a colon adenocarcinoma cell lines with wild-type and biallelic knock-in hypomorphic ATR Seckel syndrome 1 genes revealed lower cellular H 2 S levels in the mutant cells compared to the wild type [12]. ATR inhibition with the pharmacologic ATR inhibitor NU6027 also significantly lowered cellular H 2 S levels in the wild-type but not the mutant cells [12]. Treatment of both cell lines with the CBS/CSE inhibitor β-cyano-L-alanine suppressed H 2 S levels in both cell types, demonstrating that cellular H 2 S levels are regulated by CBS/CSE and ATR, which form separate regulatory foci [12].
Interestingly, ATR activation correlates with serine 435 phosphorylation, an event also required for ATR-XPA dimer formation and subsequent NER [12,80]. Treatment of the colon adenocarcinoma cell lines with β-cyano-L-alanine increased this phosphorylation, while treatment with the H 2 S donor diallyl trisulfide significantly suppressed it in the wildtype but not mutant cells. UV light and oxidative stress treatments similarly induced this phosphorylation in the wild-type but not mutant cells [12]. Activated ATR phosphorylates the CHK1 kinase serine 345, leading to its activation [1][2][3]12]. When the cells lines were pretreated with β-cyano-L-alanine, followed by a low concentration of oxidative stress, oxidative stress-induced CHK1 phosphorylation increased with H 2 S synthesis inhibition, an event again not seen in the mutant cells [12]. Lastly, to examine the effects of these events on genomic stability, oxidative stress-induced dsDNA breaks were quantified in both cell types with and without H 2 S synthesis inhibition by β-cyano-L-alanine treatment. H 2 S synthesis inhibition caused low levels of oxidative stress to significantly induce dsDNA breaks, where otherwise they were not increased [12]. The mutant cells also showed increased breaks compare to the wild-type cells [12]. Taken together, these finding indicate that ATR regulates cellular H 2 S levels and H 2 S, in turn, regulates ATR phosphorylation, ATR kinase activity, and nuclear genomic stability [12].

MEK1
An important initial and required step in the DDR is carried out by poly (ADP-ribose) polymerases (PARPS) that transfer ADP-ribose from NAD + to glutamic acid residues on a protein acceptor, creating ADP-ribose polymers at sights of DNA damage [1][2][3]81]. These chains function in the recruitment of factors involved in DNA repair such as polymerase β, XRCC1, and ligase IIIα [1][2][3]82]. PARP activation is tightly regulated by a cascade of kinases including the MEK/ERK signaling pathway [1][2][3][82][83][84]. In an interesting study, treatment of human endothelial cells for 2 h with 10 µM NaHS increased MEK1 Cys 341 S-sulfhydration. This event resulted in ERK1/2 phosphorylation and its subsequent translocation into the nucleus, where it activated PARP-1 through a direct interaction [13]. Mutation of MEK1 Cys 341 to Gly blocked these events.
Next HEK293 cells were treated with methyl methanesulfonate (MMS), which induces ssDNA and dsDNA breaks and activates PARP-1 activity, with PARP-1 then recruiting XRCC1 and DNA ligase III to initiate DNA repair [13,[83][84][85]. MMS treatment of the HEK293 cells resulted in MEK1 Cys 341 S-sulfhydration, but not in cells carrying the Gly 341 mutated MEK1 [13]. This S-sulfhydration increased in CSE over-expressing HEK293 cells. Lastly, application of MMS to human endothelial cells, with and without co-treatment with 0.1-10 µM NaHS, increased PARP-1 activity in a dose-dependent manner, with PARP-1 activation detectable at 5 min with NaHS treatment and only at 30 min without NaHS treatment [13]. NaHS treatment also reduced the amount of HEK293 cell DNA damage and increased the amounts of XRCC1 and DNA ligase III recruited [13]. This study indicates that CSE-generated H 2 S acts as a DNA damage protectant, S-sulfhydrating MEK1 Cys 341, activating ERK1/2 and PARP-1 to repair DNA damage [13].
These studies on the roles of ATR and MEK1 in nuclear DNA repair demonstrate that H 2 S plays an important and, as yet, poorly defined role in nuclear DNA repair regulation [12,13]. Interestingly, low cellular H 2 S concentrations activate ATR, as measured by its kinase activity towards CHK1 [12]. Conversely, MEK1 activity in DNA repair is increased with increased H 2 S concentrations, as supplied either by exogenous NaHS or increased CSE expression [13]. Thus, these studies imply that nuclear DNA repair is likely modulated by both increased and decreased cellular H 2 S concentrations [12,13]. Additionally, CBS and CSE knockdown both attenuated the mitochondrial interactions of EXOG with APE1, LIG3, and POL γ, implying that ATR may also indirectly regulate mitochondrial BER [11,12].

H 2 S, Autophagy, and the DDR
Recently, Jiang et al. employed a high-content screen of~12,000 with diversified chemical structures and molecular targets, screening for compounds that increased cellular H 2 S expression > 1.4 with a concomitant >50% cell survival compared to DMSO treated controls [85]. Interestingly, the largest activating compound class screened consisted of genotoxic compounds, with the most active compounds being the topoisomerase inhibitors irinotecan and teniposide, the nucleoside analog trifluridine, and bleomycin. Additionally, UVC light, ionizing radiation, and NER deficiency were also strong intracellular H 2 S inducers. Intriguingly, teniposide and UVC treatment increased the cellular sulfane sulfur fraction, indicating that the larger sulfur pool was also altered by genotoxic stress [85]. To further characterize the role of the DDR in these responses, immortalized murine embryonic fibroblasts (MEFs), with and without PARP-1 expression, were treated with UVC or teniposide [85]. PARP-1 loss significantly decreased H 2 S induction by either agent, strongly implicating a role for early DDR events in H 2 S induction [85].
Autophagy is a significant part of the DDR [86]. In MEFs knockdown of ATG5 and ATG7, two autophagy regulators, attenuated LC3I to LC3II conversion (also an autophagy marker) initiated by teniposide. Importantly, this was accompanied by a concomitant decrease in H 2 S induction, linking H 2 S induction, the DDR, and autophagy [85]. ATG5 knockout MEF viability (which showed teniposide hypersensitivity) was partially rescued from teniposide toxicity by concomitant AP39 treatment, indicating that H 2 S plays a cellular protective role in genotoxic exposure [85]. AMPK is activated by PARP-1 and is another DDR mediator [86]. In AMPK, double α1/α2 subunit knockout MEFs, H 2 S and autophagy induction with teniposide were also attenuated, again consistent with a requirement for autophagy in maximal H 2 S induction by genoclastic agents [85].
Lastly, teniposide treatment significantly induced CSE mRNA and protein in MEFs. CSE expression ablation resulted in a small but significant decrease in cellular H 2 S levels with teniposide treatment, indicating a possible role of CSE in the DDR [85]. Under genotoxic stress CSE mRNA is induced by the ATF4 transcription factor [87]. In MEFs, ATF4 expression ablation resulted in both attenuated CSE induction and lowered cellular H 2 S levels following teniposide treatment. Additionally, treatment of wild-type and ATG5 knockout MEFs with PAG decreased teniposide-induced H 2 S in both cell types. These results suggest an additive role for ATF4-mediated CSE expression and autophagy in H 2 S induction upon genotoxic stress [85]. These interactions are summarized in Figure 1.

Conclusion and Future Directions: Cancer Therapy and Cardiovascular Disease
The role of H2S in DNA repair and the DDR is an established, but as yet under researched area with few studies on the subject [11][12][13]85]. Due to the importance of genomic stability maintenance, DNA repair, and H2S in normal and pathophysiology, a better understanding of this area will undoubtedly lead to a greater understanding of the molecular pathology underlying many human diseases. For example, increased DNA Figure 1. A summary of known pathways by which H 2 S regulates mitochondrial and nuclear DNA repair integrated into one model [11][12][13]85].

Conclusions and Future Directions: Cancer Therapy and Cardiovascular Disease
The role of H 2 S in DNA repair and the DDR is an established, but as yet under researched area with few studies on the subject [11][12][13]85]. Due to the importance of genomic stability maintenance, DNA repair, and H 2 S in normal and pathophysiology, a better understanding of this area will undoubtedly lead to a greater understanding of the molecular pathology underlying many human diseases. For example, increased DNA damage in the vasculature is a major cardiovascular disease risk factor, and H 2 S is a major regulator of cardiovascular functions [14,88]. However, there is relatively little data on the role of the DDR and DNA repair in the cardiovascular system, although intriguingly murine models of defective NER show premature vascular senescence, increased vascular stiffness, and elevated blood pressure [89]. As the ATR kinase regulates NER, there may be an as yet undiscovered role for ATR in vascular diseases [1][2][3]12,89]. Additionally, the DDR protein ATM plays a poorly defined role in cardiovascular disease, with individuals and mice with only one functional ATM gene showing elevated cardiovascular disease, while a high fat diet suppresses ATM protein expression in wild-type animals [90,91]. A role for ATM in H 2 S metabolism has not yet been identified.
Increased H 2 S synthesis and CBS, CSE, and/or 3-MST expression promote cancer progression in several human malignancies, and H 2 S synthesis inhibitors have been proposed as a cancer treatment [5,11,12,17,69,92,93]. In addition, ATR inhibitors are showing promise in phase I and II clinical trials for lung, ovarian, cervical, urothelial, and advanced sold tumors [93]. Since ATR inhibition lowers cellular H 2 S concentrations, this may be an unexamined aspect of ATR inhibition in cancer therapeutics [12,92,93]. Similarly, lowered cellular H 2 S levels with ATR inhibition could, in turn, lower MEK1 and EXOG sulfhydration and activation, attenuating mitochondrial DNA repair and nuclear DNA repair mediated by the MEK1-ERK1/2-PARP-1 axis [11][12][13]. Interestingly, ATR inhibition increases the effectiveness of PARP inhibitors in cancer therapy, supporting this hypothesis [94][95][96]. Thus, H 2 S may form a molecular link uniting different aspects of DNA repair (summarized in Figure 1).
Previously, we reviewed the role of H 2 S in DNA repair with an emphasis on ATR function [69]. Here, we extended this review, as current data strongly implicate a role for other DDR proteins, especially ATM in H 2 S regulation [1][2][3][62][63][64]. Additionally, the data by Jiang et al. demonstrate that H 2 S, autophagy, and the DDR are intricately interconnected, further highlighting the function of H 2 S in the most basic functions regulating cell survival [85]. Support for this also comes from the known roles of the DDR proteins in regulating autophagy [85,97].
ATR, ATM, and DNA-PK are central to the DRR and DNA repair [1][2][3]95,96]. All three proteins share extensive sequence and substrate overlap, and synthetic lethal relationships exist between them [1][2][3]96]. Moreover, all three proteins regulate autophagy and mitochondrial function and viability [97][98][99][100]. These observations, combined with the ancient and parallel origins of H 2 S biochemistry and DNA repair, and with H 2 S now linked to mitochondrial and nuclear DNA repair, suggest that ATM and DNA-PK may also regulate aspects of H 2 S metabolism [1][2][3][11][12][13]17,[52][53][54][55][56][57][58]. Although only hypotheses, these ideas could be easily tested. In summary, the role of H 2 S in the regulation of the DDR and DNA repair is a new and exciting area of inquiry and should give useful and profound insights into normal and pathophysiology.
Author Contributions: Conceptualization and research were performed by all the authors, R.E.S., C.G.K., Y.L., and G.E.G., C.G.K. and R.E.S. wrote the main portion of the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This work supported by grants from the A-T Children's Project and an intramural research support grant from Louisiana Health Sciences Shreveport, Center for Cardiovascular Diseases and Sciences. Lastly, this work was supported by an Institutional Development Award (IDeA) from the National Institutes of General Medical Sciences of the NIH under grant numbers GM121307 and HL149264 from the National Heart, Lung, and Blood Institute to C.G.K.