Genetic investigation of formaldehyde-induced DNA damage response in Schizosaccharomyces pombe (original) (raw)

Article summary

AI generated

Formaldehyde is a prevalent environmental pollutant linked to significant health risks, including its role as a carcinogen through the formation of DNA adducts that lead to genomic instability. This study identifies the fission yeast Schizosaccharomyces pombe as a model to elucidate the genetic pathways involved in formaldehyde detoxification and DNA damage repair, revealing that the formaldehyde dehydrogenase Fmd1 is crucial for minimizing DNA lesions and that multiple repair mechanisms, including nucleotide excision repair and homologous recombination, are essential for cellular tolerance to formaldehyde-induced damage.

This is an AI-generated summary, check important information.

Abstract

Formaldehyde is a common environmental pollutant and is associated with adverse health effects. Formaldehyde is also considered to be a carcinogen because it can form DNA adducts, leading to genomic instability. How these adducts are prevented and removed is not fully understood. In this study, we used the fission yeast Schizosaccharomyces pombe as a model organism to investigate cellular tolerance pathways against formaldehyde exposure. We show that Fmd1 is a major formaldehyde dehydrogenase that functions to detoxify formaldehyde and that Fmd1 is critical to minimize formaldehyde-mediated DNA lesions. Our investigation revealed that nucleotide excision repair and homologous recombination have major roles in cellular tolerance to formaldehyde, while mutations in the Fanconi anemia, translesion synthesis, and base excision repair pathways also render cells sensitive to formaldehyde. We also demonstrate that loss of Wss1 or Wss2, proteases involved in the removal of DNA–protein crosslinks, sensitizes cells to formaldehyde and leads to replication defects. These results suggest that formaldehyde generates a variety of DNA lesions, including interstrand crosslinks, DNA–protein crosslinks, and base adducts. Thus, our genetic studies provide a framework for future investigation regarding health effects resulting from formaldehyde exposure.

Similar content being viewed by others

Introduction

Formaldehyde is commonly used in industrial and consumer products, raising occupational and non-occupational health risks. Formaldehyde is also a major environmental pollutant produced by fires, cigarettes, and automotive exhaust (Kawanishi et al. 2014). Since 1980, when formaldehyde was found to induce squamous cell carcinoma in the rat nasal cavity (Swenberg et al. 1980), numerous epidemiological studies have demonstrated roles of formaldehyde in carcinogenesis (Swenberg et al. [2013](/article/10.1007/s00294-020-01057-z#ref-CR50 "Swenberg JA, Moeller BC, Lu K, Rager JE, Fry RC, Starr TB (2013) Formaldehyde carcinogenicity research: 30 years and counting for mode of action, epidemiology, and cancer risk assessment. Toxicol Pathol 41:181–189. https://doi.org/10.1177/0192623312466459

")). Accordingly, formaldehyde was classified as a human carcinogen at IARC 2004 (International Agency for Research on Cancer) (IARC 2004).

Carcinogenic effects of formaldehyde are probably due to its role in modifying DNA. Formaldehyde reacts with deoxynucleotides, forming base adducts including _N_6-hydroxymethyldeoxyadenosine, _N_4-hydroxymethyldeoxycytidine, and _N_2-hydroxymethyldeoxyguanosine, which could be removed by the base excision repair (BER) pathway (Fig. 1a) (Kawanishi et al. 2014). Formaldehyde also induces DNA–protein crosslinks (DPCs) (Klages-Mundt and Li [2017](/article/10.1007/s00294-020-01057-z#ref-CR21 "Klages-Mundt NL, Li L (2017) Formation and repair of DNA-protein crosslink damage. Sci China Life Sci 60:1065–1076. https://doi.org/10.1007/s11427-017-9183-4

")). Such bulky DNA lesions appear to induce nucleotide excision repair (NER) or homologous recombination (HR), depending on the size of proteins crosslinked onto DNA (Kawanishi et al. 2014). Studies demonstrated in vitro that HR-mediated fork cleavage at DPCs plays a critical role in mammalian DPC repair if the crosslinked proteins are larger than 8–10 kDa (Nakano et al. [2009](/article/10.1007/s00294-020-01057-z#ref-CR30 "Nakano T, Katafuchi A, Matsubara M, Terato H, Tsuboi T, Masuda T, Tatsumoto T, Pack SP, Makino K, Croteau DL, Van Houten B, Iijima K, Tauchi H, Ide H (2009) Homologous recombination but not nucleotide excision repair plays a pivotal role in tolerance of DNA-protein cross-links in mammalian cells. J Biol Chem 284:27065–27076. https://doi.org/10.1074/jbc.M109.019174

")). However, studies also showed that NER still processes DPCs with relatively small proteins that are less than 12–14 kDa (Nakano et al. [2007](/article/10.1007/s00294-020-01057-z#ref-CR29 "Nakano T, Morishita S, Katafuchi A, Matsubara M, Horikawa Y, Terato H, Salem AM, Izumi S, Pack SP, Makino K, Ide H (2007) Nucleotide excision repair and homologous recombination systems commit differentially to the repair of DNA-protein crosslinks. Mol Cell 28:147–158. https://doi.org/10.1016/j.molcel.2007.07.029

")). Therefore, while the majority of DPCs undergo HR-mediated DNA repair, NER also plays an important role in DPC repair (Fig. 1a). Consistently, a formaldehyde sensitivity screen in Saccharomyces cerevisiae also identified multiple proteins involved in HR and NER (de Graaf et al. [2009](/article/10.1007/s00294-020-01057-z#ref-CR11 "de Graaf B, Clore A, McCullough AK (2009) Cellular pathways for DNA repair and damage tolerance of formaldehyde-induced DNA-protein crosslinks. DNA Repair 8:1207–1214. https://doi.org/10.1016/j.dnarep.2009.06.007

")), further strengthening the role of HR and NER in cellular tolerance to formaldehyde.

Fig. 1

Fig. 1

The alternative text for this image may have been generated using AI.

Full size image

Genes involved in cellular tolerance to formaldehyde. a Fmd1, Adh1, and Atd1 detoxify formaldehyde. Formaldehyde induces a variety of DNA lesions including base adducts, DPCs and ICLs, which activate different types of DNA repair pathways. b–d Five-fold serial dilutions of the indicated cells were plated on YES agar medium containing the indicated amounts of formaldehyde or acetaldehyde. Cells were then incubated for 3–5 days at 30 °C in a sealed condition to minimize aldehyde evaporation. The initial concentrations of aldehydes are shown. Representative images of repeat experiments are shown

Recent studies have added another layer of understanding of the mechanism of DPC repair. In S. cerevisiae, the metalloprotease Wss1 promotes proteolytic digestion of proteins crosslinked onto DNA in response to formaldehyde (Fig. 1a) (Stingele et al. [2014](/article/10.1007/s00294-020-01057-z#ref-CR46 "Stingele J, Schwarz MS, Bloemeke N, Wolf PG, Jentsch S (2014) A DNA-dependent protease involved in DNA-protein crosslink repair. Cell 158:327–338. https://doi.org/10.1016/j.cell.2014.04.053

")). Similarly, Spartan/DVC1, the mammalian homolog of Wss1, was identified as a DNA replication-coupled protease that promotes DPC repair (Lopez-Mosqueda et al. 2016; Stingele et al. [2016](/article/10.1007/s00294-020-01057-z#ref-CR47 "Stingele J, Bellelli R, Alte F, Hewitt G, Sarek G, Maslen SL, Tsutakawa SE, Borg A, Kjaer S, Tainer JA, Skehel JM, Groll M, Boulton SJ (2016) Mechanism and regulation of DNA-protein crosslink repair by the DNA-dependent metalloprotease SPRTN. Mol Cell 64:688–703. https://doi.org/10.1016/j.molcel.2016.09.031

"); Vaz et al. [2016](/article/10.1007/s00294-020-01057-z#ref-CR51 "Vaz B, Popovic M, Newman JA, Fielden J, Aitkenhead H, Halder S, Singh AN, Vendrell I, Fischer R, Torrecilla I, Drobnitzky N, Freire R, Amor DJ, Lockhart PJ, Kessler BM, McKenna GW, Gileadi O, Ramadan K (2016) Metalloprotease SPRTN/DVC1 orchestrates replication-coupled DNA-protein crosslink repair. Mol Cell 64:704–719. https://doi.org/10.1016/j.molcel.2016.09.032

")). Such proteolytic processing may leave a short peptide still crosslinked onto DNA (Duxin et al. [2014](/article/10.1007/s00294-020-01057-z#ref-CR12 "Duxin JP, Dewar JM, Yardimci H, Walter JC (2014) Repair of a DNA-protein crosslink by replication-coupled proteolysis. Cell 159:346–357. https://doi.org/10.1016/j.cell.2014.09.024

")); however, by reducing the size of crosslinked proteins, Wss1/Spartan/DVC1 proteases may facilitate efficient removal of DNA adducts by NER or other DNA repair mechanisms (Fig. 1a).

Although DPCs may represent a majority of formaldehyde-induced DNA lesions, formaldehyde is also known to form other DNA adducts, which complicates the interpretation of formaldehyde genotoxicity (Klages-Mundt and Li [2017](/article/10.1007/s00294-020-01057-z#ref-CR21 "Klages-Mundt NL, Li L (2017) Formation and repair of DNA-protein crosslink damage. Sci China Life Sci 60:1065–1076. https://doi.org/10.1007/s11427-017-9183-4

")). These adducts include interstrand crosslinks (ICLs) and base adducts. ICLs are mainly repaired by the Fanconi anemia (FA) DNA repair pathway (Fig. 1a) (Ceccaldi et al. [2016](/article/10.1007/s00294-020-01057-z#ref-CR7 "Ceccaldi R, Sarangi P, D'Andrea AD (2016) The Fanconi anaemia pathway: new players and new functions. Nat Rev Mol Cell Biol 17:337–349. https://doi.org/10.1038/nrm.2016.48

")). Indeed, mammalian cells deficient in the FA protein FANCD2 display sensitivity to formaldehyde (Pontel et al. [2015](/article/10.1007/s00294-020-01057-z#ref-CR39 "Pontel LB, Rosado IV, Burgos-Barragan G, Garaycoechea JI, Yu R, Arends MJ, Chandrasekaran G, Broecker V, Wei W, Liu L, Swenberg JA, Crossan GP, Patel KJ (2015) Endogenous formaldehyde is a hematopoietic stem cell genotoxin and metabolic carcinogen. Mol Cell 60:177–188. https://doi.org/10.1016/j.molcel.2015.08.020

"); Ren et al. [2013](/article/10.1007/s00294-020-01057-z#ref-CR41 "Ren X, Ji Z, McHale CM, Yuh J, Bersonda J, Tang M, Smith MT, Zhang L (2013) The impact of FANCD2 deficiency on formaldehyde-induced toxicity in human lymphoblastoid cell lines. Arch Toxicol 87:189–196. https://doi.org/10.1007/s00204-012-0911-6

")). Furthermore, it is highly possible that formaldehyde-induced base adducts are repaired by the base excision repair (BER) pathway (Luch et al. [2014](/article/10.1007/s00294-020-01057-z#ref-CR26 "Luch A, Frey FC, Meier R, Fei J, Naegeli H (2014) Low-dose formaldehyde delays DNA damage recognition and DNA excision repair in human cells. PLoS ONE 9:e94149. https://doi.org/10.1371/journal.pone.0094149

")).

Taken together, previous studies have reported that formaldehyde activates a variety of DNA repair pathways (Fig. 1a). However, these studies are done in various cell lines with different genetic profiles, making the interpretation of formaldehyde toxicity very difficult. Therefore, in this report, we dissected genetic pathways involved in formaldehyde tolerance by using a model organism, the fission yeast Schizosaccharomyces pombe, in which nearly all laboratory strains are isogenic (Forsburg and Rhind [2006](/article/10.1007/s00294-020-01057-z#ref-CR14 "Forsburg SL, Rhind N (2006) Basic methods for fission yeast. Yeast 23:173–183. https://doi.org/10.1002/yea.1347

"); Hoffman et al. [2015](/article/10.1007/s00294-020-01057-z#ref-CR16 "Hoffman CS, Wood V, Fantes PA (2015) An ancient yeast for young geneticists: a primer on the Schizosaccharomyces pombe model system. Genetics 201:403–423. https://doi.org/10.1534/genetics.115.181503

"); Wood et al. [2002](/article/10.1007/s00294-020-01057-z#ref-CR52 "Wood V, Gwilliam R, Rajandream MA, Lyne M, Lyne R, Stewart A, Sgouros J, Peat N, Hayles J, Baker S, Basham D, Bowman S, Brooks K, Brown D, Brown S, Chillingworth T, Churcher C, Collins M, Connor R, Cronin A, Davis P, Feltwell T, Fraser A, Gentles S, Goble A, Hamlin N, Harris D, Hidalgo J, Hodgson G, Holroyd S, Hornsby T, Howarth S, Huckle EJ, Hunt S, Jagels K, James K, Jones L, Jones M, Leather S, McDonald S, McLean J, Mooney P, Moule S, Mungall K, Murphy L, Niblett D, Odell C, Oliver K, O'Neil S, Pearson D, Quail MA, Rabbinowitsch E, Rutherford K, Rutter S, Saunders D, Seeger K, Sharp S, Skelton J, Simmonds M, Squares R, Squares S, Stevens K, Taylor K, Taylor RG, Tivey A, Walsh S, Warren T, Whitehead S, Woodward J, Volckaert G, Aert R, Robben J, Grymonprez B, Weltjens I, Vanstreels E, Rieger M, Schafer M, Muller-Auer S, Gabel C, Fuchs M, Dusterhoft A, Fritzc C, Holzer E, Moestl D, Hilbert H, Borzym K, Langer I, Beck A, Lehrach H, Reinhardt R, Pohl TM, Eger P, Zimmermann W, Wedler H, Wambutt R, Purnelle B, Goffeau A, Cadieu E, Dreano S, Gloux S, Lelaure V, Mottier S, Galibert F, Aves SJ, Xiang Z, Hunt C, Moore K, Hurst SM, Lucas M, Rochet M, Gaillardin C, Tallada VA, Garzon A, Thode G, Daga RR, Cruzado L, Jimenez J, Sanchez M, del Rey F, Benito J, Dominguez A, Revuelta JL, Moreno S, Armstrong J, Forsburg SL, Cerutti L, Lowe T, McCombie WR, Paulsen I, Potashkin J, Shpakovski GV, Ussery D, Barrell BG, Nurse P (2002) The genome sequence of Schizosaccharomyces pombe. Nature 415:871–880. https://doi.org/10.1038/nature724

")). We show that the Fmd1 formaldehyde dehydrogenase prevents DNA damage upon formaldehyde exposure. We also provide genetic evidence that formaldehyde generates a variety of DNA adducts, including ICLs, DPCs, and base adducts. Formaldehyde appears to cause DNA replication stress, activating fork protection and cell cycle checkpoint pathways. Our investigation revealed that NER and HR have major roles in the repair of formaldehyde-induced DNA lesions, while BER, the FA pathway, and translesion synthesis (TLS) also contribute to the cellular tolerance to formaldehyde. We will also discuss differences in DNA repair response against formaldehyde and acetaldehyde, the latter of which is the primary metabolite of ethanol and another human carcinogen (Brooks and Zakhari [2014](/article/10.1007/s00294-020-01057-z#ref-CR5 "Brooks PJ, Zakhari S (2014) Acetaldehyde and the genome: beyond nuclear DNA adducts and carcinogenesis. Environ Mol Mutagen 55:77–91. https://doi.org/10.1002/em.21824

")). Thus, our studies will contribute to the development of further hypotheses, building towards future investigations into the mechanisms of the aldehyde-induced DNA damage response.

Materials and methods

General techniques and S. pombe strains

The methods for genetic and molecular biology analyses of fission yeast have been described previously (Alfa et al. 1993; Moreno et al. 1991). The S. pombe strains used in this study were generated using standard techniques, and their genotypes are listed in Supplementary Table S1.

_fmd1_∆ (fmd1::hphMX6) was constructed by a PCR-based method (Krawchuk and Wahls [1999](/article/10.1007/s00294-020-01057-z#ref-CR22 "Krawchuk MD, Wahls WP (1999) High-efficiency gene targeting in Schizosaccharomyces pombe using a modular, PCR-based approach with long tracts of flanking homology. Yeast 15:1419–1427. https://doi.org/10.1002/(sici)1097-0061(19990930)15:13%3c1419:Aid-yea466%3e3.0.Co;2-q

")) to replace the open reading frame with the hphMX6 gene. Mutations have been described for _adh1_∆ (adh1::kanMX6) (Corkins et al. [2013](/article/10.1007/s00294-020-01057-z#ref-CR9 "Corkins ME, May M, Ehrensberger KM, Hu YM, Liu YH, Bloor SD, Jenkins B, Runge KW, Bird AJ (2013) Zinc finger protein Loz1 is required for zinc-responsive regulation of gene expression in fission yeast. Proc Natl Acad Sci USA 110:15371–15376. https://doi.org/10.1073/pnas.1300853110

")), _rad3_∆ (rad3::kanMX4), _chl1_∆ (chl1::hphMX6) (Ansbach et al. [2008](/article/10.1007/s00294-020-01057-z#ref-CR3 "Ansbach AB, Noguchi C, Klansek IW, Heidlebaugh M, Nakamura TM, Noguchi E (2008) RFCCtf18 and the Swi1-Swi3 complex function in separate and redundant pathways required for the stabilization of replication forks to facilitate sister chromatid cohesion in Schizosaccharomyces pombe. Mol Biol Cell 19:595–607. https://doi.org/10.1091/mbc.E07-06-0618

")), _swi1_∆ (swi1::kanMX6) (Noguchi et al. 2003), _rad51_∆ (rad51::_kan_r) (Lambert et al. [2005](/article/10.1007/s00294-020-01057-z#ref-CR23 "Lambert S, Watson A, Sheedy DM, Martin B, Carr AM (2005) Gross chromosomal rearrangements and elevated recombination at an inducible site-specific replication fork barrier. Cell 121:689–702. https://doi.org/10.1016/j.cell.2005.03.022

")), _rad52_∆ (rad52::hphMX6) (Gadaleta et al. [2016](/article/10.1007/s00294-020-01057-z#ref-CR15 "Gadaleta MC, Das MM, Tanizawa H, Chang YT, Noma K, Nakamura TM, Noguchi E (2016) Swi1timeless prevents repeat instability at fission yeast telomeres. PLoS Genet 12:e1005943. https://doi.org/10.1371/journal.pgen.1005943

")), _atd2_∆ (atd2::hphMX6), _fmd1_∆ (fmd1::hphMX6), _wss1_∆ (wss1::hphMX6), _wss2_∆ (wss2::kanMX6) (Noguchi et al. [2017](/article/10.1007/s00294-020-01057-z#ref-CR36 "Noguchi C, Grothusen G, Anandarajan V, Martinez-Lage Garcia M, Terlecky D, Corzo K, Tanaka K, Nakagawa H, Noguchi E (2017) Genetic controls of DNA damage avoidance in response to acetaldehyde in fission yeast. Cell Cycle 16:45–58. https://doi.org/10.1080/15384101.2016.1237326

")), _fml1_∆ (fml1::natMX6) (Nandi and Whitby [2012](/article/10.1007/s00294-020-01057-z#ref-CR31 "Nandi S, Whitby MC (2012) The ATPase activity of Fml1 is essential for its roles in homologous recombination and DNA repair. Nucleic Acids Res 40:9584–9595. https://doi.org/10.1093/nar/gks715

")), _rad26_∆ (rad26::ura4+) (al-Khodairy, et al. 1994), _nth1_∆ (nth1::ura4+), _apn2_∆ (apn2::ura4+) (Sugimoto et al. [2005](/article/10.1007/s00294-020-01057-z#ref-CR48 "Sugimoto T, Igawa E, Tanihigashi H, Matsubara M, Ide H, Ikeda S (2005) Roles of base excision repair enzymes Nth1p and Apn2p from Schizosaccharomyces pombe in processing alkylation and oxidative DNA damage. DNA Repair 4:1270–1280. https://doi.org/10.1016/j.dnarep.2005.06.009

")), _swi9_∆ (swi9::ura4+) (Carr et al. 1994), _swi10_∆ (swi10::ura4+) (Rodel et al. 1992), _rad3_∆ (rad3::ura4+) (Baber-Furnari et al. 2000), _eso1_∆ (eso1::kanMX6), _kap1_∆ (kap1::bleMX6), and _rev3_∆ (rev3::hphMX6) (Sheedy et al. [2005](/article/10.1007/s00294-020-01057-z#ref-CR45 "Sheedy DM, Dimitrova D, Rankin JK, Bass KL, Lee KM, Tapia-Alveal C, Harvey SH, Murray JM, O'Connell MJ (2005) Brc1-mediated DNA repair and damage tolerance. Genetics 171:457–468. https://doi.org/10.1534/genetics.105.044966

")). _fmd1_∆ (fmd1::kanMX4), _fmd2_∆ (fmd2::kanMX4), _fmd3_∆ (fmd3::kanMX4), _atd1_∆ (atd1::kanMX4), _atd2_∆ (atd2::kanMX4), _atd3_∆ (atd3::kanMX4), _pso2_∆ (pso2::kanMX4), and _fan1_∆ (fan1::kanMX4) cells were obtained rom Bioneer, Inc.

Drug sensitivity assays

Formaldehyde and acetaldehyde sensitivity assays were performed as described (Noguchi et al. [2017](/article/10.1007/s00294-020-01057-z#ref-CR36 "Noguchi C, Grothusen G, Anandarajan V, Martinez-Lage Garcia M, Terlecky D, Corzo K, Tanaka K, Nakagawa H, Noguchi E (2017) Genetic controls of DNA damage avoidance in response to acetaldehyde in fission yeast. Cell Cycle 16:45–58. https://doi.org/10.1080/15384101.2016.1237326

")). Acetaldehyde (402,788, ≥ 99.5%) and formaldehyde (F8775, 36.5–38%) were purchased from Sigma-Aldrich (St. Louis, MO). Both aldehydes are highly volatile. Therefore, to minimize evaporation, aldehyde solution and aldehyde-containing agar medium were prepared on ice or in a room maintained at 4 °C.

Serial dilution growth assays were performed on aldehyde-containing plates using exponentially growing S. pombe cells. Plates were sealed with Parafilm, incubated at 32 °C for 3–5 days to allow cell growth, and photographed. Because evaporation of acetaldehyde cannot be avoided, the initial concentrations of aldehyde used in each experiment are indicated.

Detection of Rad52-YFP DNA repair foci

To detect Rad52-YFP foci, pJK210-Rad52CT-YFP (Noguchi et al. [2012](/article/10.1007/s00294-020-01057-z#ref-CR35 "Noguchi C, Rapp JB, Skorobogatko YV, Bailey LD, Noguchi E (2012) Swi1 associates with chromatin through the DDT domain and recruits Swi3 to preserve genomic integrity. PLoS ONE 7:e43988. https://doi.org/10.1371/journal.pone.0043988

"); Rapp et al. [2010](/article/10.1007/s00294-020-01057-z#ref-CR40 "Rapp JB, Noguchi C, Das MM, Wong LK, Ansbach AB, Holmes AM, Arcangioli B, Noguchi E (2010) Checkpoint-dependent and -independent roles of Swi3 in replication fork recovery and sister chromatid cohesion in fission yeast. PLoS ONE 5:e13379. https://doi.org/10.1371/journal.pone.0013379

")) was digested by _Alf_II and inserted at the rad52 locus of S. pombe strains. Cells expressing Rad52-YFP were incubated in regular growth medium until mid-log phase at 25 °C. This temperature was used to preserve YFP fluorescence and to reduce background signal. Cells were treated with formaldehyde or acetaldehyde for 2–3 h in a test tube sealed with Parafilm to minimize their evaporation. Live-cell imaging analysis of Rad52-YFP was performed as described (Noguchi et al. [2009](/article/10.1007/s00294-020-01057-z#ref-CR34 "Noguchi E, Ansbach AB, Noguchi C, Russell P (2009) Assays used to study the DNA replication checkpoint in fission yeast. Methods Mol Biol 521:493–507. https://doi.org/10.1007/978-1-60327-815-7_28

")).

Results

Genes involved in formaldehyde detoxification in S. pombe

To understand defense mechanisms against formaldehyde exposure, we performed bioinformatics analysis to identify genes that are closely related to the glutathione-dependent formaldehyde dehydrogenase ADH5, the main enzyme for formaldehyde detoxification in humans (Iborra et al. [1992](/article/10.1007/s00294-020-01057-z#ref-CR18 "Iborra FJ, Renau-Piqueras J, Portoles M, Boleda MD, Guerri C, Pares X (1992) Immunocytochemical and biochemical demonstration of formaldhyde dehydrogenase (class III alcohol dehydrogenase) in the nucleus. J Histochem Cytochem 40:1865–1878. https://doi.org/10.1177/40.12.1453005

"); Rosado et al. [2011](/article/10.1007/s00294-020-01057-z#ref-CR43 "Rosado IV, Langevin F, Crossan GP, Takata M, Patel KJ (2011) Formaldehyde catabolism is essential in cells deficient for the Fanconi anemia DNA-repair pathway. Nat Struct Mol Biol 18:1432–1434. https://doi.org/10.1038/nsmb.2173

")). The SPCC13B11.04c protein showed 64% identity and 76% similarity to human ADH5 (query coverage, 99%). The SPBC1539.07c protein also showed a high similarity to ADH5 (61% identity and 74% similarity over 99% of the ADH5 query amino acid sequence). There are additional proteins that were related to ADH5. These include alcohol dehydrogenase Adh1 and SPBC1198.01, which have 33% and 27% identity to ADH5, respectively. Based on their sequence similarities to formaldehyde dehydrogenase, SPBC1539.07c, SPBC1198.01, and SPCC13B11.04c were designated fmd1, fmd2, and fmd3 (putative ForMaldehyde Dehydrogenase). These annotations have been accepted by and are available from Pombase (https://www.pombase.org), the fission yeast database.

To determine whether these genes are required for formaldehyde detoxification in S. pombe, we examined formaldehyde sensitivity of cells deleted of fmd1, fmd2, or fmd3. Formaldehyde is volatile; it is difficult to control its concentration in the growth medium. In addition, formaldehyde sensitivity also depends on its freshness. Therefore, formaldehyde sensitivity of the same strains varies in different experiments; however, the relative levels of sensitivity among different mutants show similar trends. As shown in Fig. 1b, _fmd1_∆ cells showed hypersensitivity to formaldehyde, whereas _fmd2_∆ and _fmd3_∆ cells failed to show formaldehyde sensitivity. Because Adh1 alcohol dehydrogenase also shows a similarity to human ADH5 at the amino acid sequence level, we also checked the formaldehyde sensitivity of cells deleted for the adh1 gene. Accordingly, _adh1_∆ cells were tested for formaldehyde sensitivity. We found that _adh1_∆ cells are hypersensitive to formaldehyde (Fig. 1c). Interestingly, _adh1_∆ cells showed stronger formaldehyde sensitivity when compared to _fmd1_∆ cells (Fig. 1c), indicating that alcohol dehydrogenase Adh1 also plays a major role in formaldehyde resistance.

To investigate functional specificity of Fmd1 in formaldehyde detoxification, we also determined whether Fmd1 is involved in detoxification of acetaldehyde, another toxic aldehydic substance. For this purpose, we compared acetaldehyde sensitivity of _fmd1_∆ cells with that of _atd1_∆, _atd2_∆, and _atd3_∆ cells, which have deficiency in acetaldehyde dehydrogenase genes (Noguchi et al. [2017](/article/10.1007/s00294-020-01057-z#ref-CR36 "Noguchi C, Grothusen G, Anandarajan V, Martinez-Lage Garcia M, Terlecky D, Corzo K, Tanaka K, Nakagawa H, Noguchi E (2017) Genetic controls of DNA damage avoidance in response to acetaldehyde in fission yeast. Cell Cycle 16:45–58. https://doi.org/10.1080/15384101.2016.1237326

")). Interestingly, _fmd1_∆ cells were not sensitive to acetaldehyde (Fig. 1b). In contrast, _atd1_∆ cells were sensitive to both acetaldehyde and formaldehyde (Fig. 1b). In addition, _adh1_∆ cells also showed acetaldehyde sensitivity (Fig. 1d). Taken together, we concluded that Fmd1 has critical roles in formaldehyde detoxification, whereas Atd1 and Adh1 function to detoxify both formaldehyde and acetaldehyde.

Fmd1, Atd1, and Adh1 prevent further accumulation of DNA damage in response to formaldehyde.

Next, we investigated whether formaldehyde treatment results in DNA damage in S. pombe cells. For this purpose, S. pombe cells were engineered to express the Rad52 DNA repair protein fused to yellow fluorescent protein (Rad52-YFP). Rad52 is involved in homologous recombination and forms DNA repair foci at DNA damage sites (Noguchi et al. 2003). Formaldehyde caused significantly elevated levels of Rad52-YFP foci in wild-type cells, indicating that formaldehyde causes DNA damage (Fig. 2a, b). Importantly, _fmd1_∆, _atd1_∆, and _adh1_∆ cells further accumulated Rad52-YFP DNA repair foci in response to formaldehyde. _fmd1_∆ and _adh1_∆ cells had considerably higher levels of Rad52 foci than _atd1_∆ cells (Fig. 2 a,b, Supplementary Fig 1a). Consistent with these results, _rad52_∆ cells were highly sensitive to formaldehyde (Fig. 2c), suggesting that Rad52-dependent DNA repair pathway is activated in response to formaldehyde. Taken together, we concluded that formaldehyde induces DNA damage and that Fmd1, Atd1, and Adh1 prevent further accumulation of formaldehyde-induced DNA damage.

Fig. 2

Fig. 2

The alternative text for this image may have been generated using AI.

Full size image

Fmd1 and Atd1 prevent accumulation of formaldehyde-mediated DNA damage. a Rad52-YFP expressing cells of the indicated genotypes were grown to mid-log phase at 25 °C and treated with 0.5 mM formaldehyde for 2 h. Percentages of nuclei with at least one Rad52-YFP focus and 2 or more foci are shown. At least 200 cells were counted for each strain. Error bars correspond to standard errors of mean (SEM) obtained from 3 independent experiments. Student’s _t_-test, *p < 0.05, ***p < 0.001. b Representative images of the formaldehyde-treated cells in A are shown. c Five-fold dilutions of wild-type, _rad51_∆, and _rad52_∆ cells were plated on YES agar medium containing the indicated concentrations of formaldehyde. Cells were then incubated for 3–5 days at 32 °C. d Rad52-YFP-expressing cells of the indicated genotypes were treated with 10 mM acetaldehyde for 3 h. Rad52-YFP foci analysis was performed as described in A. Student’s _t_-test, **p < 0.01, ns not significant

As described above, _atd1_∆ and _adh1_∆ cells showed sensitivity to both formaldehyde and acetaldehyde, whereas _fmd1_∆ cells were only sensitive to formaldehyde (Fig. 1). This suggests that Fmd1 functions to specifically detoxify formaldehyde. To test this possibility, we monitored Rad52-YFP foci formation in acetaldehyde-treated _fmd1_∆ cells (Fig. 2d). As a control, we used _atd2_∆ cells because we previously showed that both _atd1_∆ and _atd2_∆ cells are important for acetaldehyde detoxification. Although _atd2_∆ cells are not significantly sensitive to acetaldehyde (Fig. 1a), _atd1_∆ _atd2_∆ double mutant cells are much more sensitive to acetaldehyde than either single mutant cells, suggesting that Atd2 has a minor role in acetaldehyde detoxification (Noguchi et al. [2017](/article/10.1007/s00294-020-01057-z#ref-CR36 "Noguchi C, Grothusen G, Anandarajan V, Martinez-Lage Garcia M, Terlecky D, Corzo K, Tanaka K, Nakagawa H, Noguchi E (2017) Genetic controls of DNA damage avoidance in response to acetaldehyde in fission yeast. Cell Cycle 16:45–58. https://doi.org/10.1080/15384101.2016.1237326

")). Consistently, we previously showed that _atd2_∆ cells display elevated levels of Rad52 foci formation, which is also confirmed in Fig. 2d. In contrast, acetaldehyde failed to cause further accumulation of DNA damage in _fmd1_∆ cells when compared to wild-type cells (Fig. 2d), suggesting that Fmd1’s function is more specific to formaldehyde detoxification.

Although it is interesting to further investigate the roles of various dehydrogenases in tolerance to different aldehydes, the focus of this study is to understand genomic instability in response to formaldehyde. Accordingly, in this report, we will further describe the roles of Fmd1 and cellular responses to formaldehyde.

Formaldehyde induces the DNA replication checkpoint

DNA damage induced by formaldehyde should activate cell cycle checkpoints to allow time for DNA repair. Therefore, we tested formaldehyde sensitivity of _rad3_∆ and _rad26_∆ cells. S. pombe Rad3 is a homolog of the ATR checkpoint kinase and forms a complex with Rad26 (ATRIP homolog) to function as a key player in the cell cycle checkpoint signaling in response to replication stress (known as the DNA replication checkpoint, Fig. 3a) (Corcoles-Saez et al. [2019](/article/10.1007/s00294-020-01057-z#ref-CR8 "Corcoles-Saez I, Dong K, Cha RS (2019) Versatility of the Mec1(ATM/ATR) signaling network in mediating resistance to replication, genotoxic, and proteotoxic stresses. Curr Genet 65:657–661. https://doi.org/10.1007/s00294-018-0920-y

"); Hustedt et al. [2013](/article/10.1007/s00294-020-01057-z#ref-CR17 "Hustedt N, Gasser SM, Shimada K (2013) Replication checkpoint: tuning and coordination of replication forks in s phase. Genes 4:388–434. https://doi.org/10.3390/genes4030388

"); Moriel-Carretero et al. [2019](/article/10.1007/s00294-020-01057-z#ref-CR28 "Moriel-Carretero M, Pasero P, Pardo B (2019) DDR Inc., one business, two associates. Curr Genet 65:445–451. https://doi.org/10.1007/s00294-018-0908-7

"); Noguchi 2010). Both _rad3_∆ and _rad26_∆ cells showed considerable formaldehyde sensitivity (Fig. 3b,c). In addition, _rad3_∆ _fmd1_∆ double mutant cells displayed stronger formaldehyde sensitivity than either single mutant cells (Fig. 3b), suggesting that DNA damage induced by formaldehyde activates the DNA replication checkpoint.

Fig. 3

Fig. 3

The alternative text for this image may have been generated using AI.

Full size image

Formaldehyde induces replication stress. a Formaldehyde incudes DPCs that interfere with replication fork progression, leading to activation of the ATR-ATRIP-dependent DNA replication checkpoint pathway to arrest the cell cycle. Swi1, a component of the replication fork protection complex, plays a critical role in stabilization of replication fork structures, while Wss1 and Wss2 proteases digest proteins crosslinked onto DNA. b–d Formaldehyde sensitivity of the indicated cells was evaluated as described in Fig. 1. For each condition, all strains were plated on the same plate for formaldehyde or acetaldehyde sensitivity assay. Replication stress checkpoint (_swi1_∆, _rad3_∆, and _rad26_∆) and FA mutants (_fml1_∆ and _chl1_∆) are sensitive to formaldehyde

We previously demonstrated that loss of Swi1Timeless, a protein critical for replication fork stabilization in response to DNA damage (Noguchi et al. 2003), sensitizes S. pombe cells to acetaldehyde, and that acetaldehyde induces DNA damage during DNA replication (Noguchi et al. [2017](/article/10.1007/s00294-020-01057-z#ref-CR36 "Noguchi C, Grothusen G, Anandarajan V, Martinez-Lage Garcia M, Terlecky D, Corzo K, Tanaka K, Nakagawa H, Noguchi E (2017) Genetic controls of DNA damage avoidance in response to acetaldehyde in fission yeast. Cell Cycle 16:45–58. https://doi.org/10.1080/15384101.2016.1237326

")). _swi1_∆ cells displayed mild sensitivity to formaldehyde (Fig. 3c); however, as reported previously (Ansbach et al. [2008](/article/10.1007/s00294-020-01057-z#ref-CR3 "Ansbach AB, Noguchi C, Klansek IW, Heidlebaugh M, Nakamura TM, Noguchi E (2008) RFCCtf18 and the Swi1-Swi3 complex function in separate and redundant pathways required for the stabilization of replication forks to facilitate sister chromatid cohesion in Schizosaccharomyces pombe. Mol Biol Cell 19:595–607. https://doi.org/10.1091/mbc.E07-06-0618

"); Noguchi et al. 2003), these cells were hypersensitive to replication-stressing agents including hydroxyurea (HU) and camptothecin (CPT) (Supplementary Fig S1B). In contrast, _fmd1_∆ cells failed to show sensitivity to HU or CPT (Supplementary Fig S1B), suggesting that Fmd1 is not directly involved in checkpoint activation in response to replication stress. Importantly, formaldehyde sensitivity of _swi1_∆ cells was further enhanced by fmd1 deletion as _swi1_∆ _fmd1_∆ cells were more sensitive to formaldehyde than either single mutant cells (Fig. 3d), suggesting that formaldehyde causes replication fork instability. Furthermore, _swi1_∆ _rad26_∆ cells were more sensitive to formaldehyde than either single mutant cells (Fig. 3c). Considering that the Rad3ATR-Rad26ATRIP complex exerts checkpoint signaling in response to DNA damage during DNA replication (Hustedt et al. [2013](/article/10.1007/s00294-020-01057-z#ref-CR17 "Hustedt N, Gasser SM, Shimada K (2013) Replication checkpoint: tuning and coordination of replication forks in s phase. Genes 4:388–434. https://doi.org/10.3390/genes4030388

"); Noguchi 2010), our results are consistent with the notion that formaldehyde induces DNA damage during DNA replication.

Involvement of DPC proteases in preventing DNA damage in response to formaldehyde

Formaldehyde is often used as a DPC-inducing agent (Klages-Mundt and Li [2017](/article/10.1007/s00294-020-01057-z#ref-CR21 "Klages-Mundt NL, Li L (2017) Formation and repair of DNA-protein crosslink damage. Sci China Life Sci 60:1065–1076. https://doi.org/10.1007/s11427-017-9183-4

")). DPCs appear to be removed by a DNA replication-associated mechanism that utilizes the Wss1/Spartan/DVC1 protease required for digestion of crosslinked proteins (Fig. 3a) (Lopez-Mosqueda et al. 2016; Stingele et al. [2016](/article/10.1007/s00294-020-01057-z#ref-CR47 "Stingele J, Bellelli R, Alte F, Hewitt G, Sarek G, Maslen SL, Tsutakawa SE, Borg A, Kjaer S, Tainer JA, Skehel JM, Groll M, Boulton SJ (2016) Mechanism and regulation of DNA-protein crosslink repair by the DNA-dependent metalloprotease SPRTN. Mol Cell 64:688–703. https://doi.org/10.1016/j.molcel.2016.09.031

"), [2014](/article/10.1007/s00294-020-01057-z#ref-CR46 "Stingele J, Schwarz MS, Bloemeke N, Wolf PG, Jentsch S (2014) A DNA-dependent protease involved in DNA-protein crosslink repair. Cell 158:327–338. https://doi.org/10.1016/j.cell.2014.04.053

"); Vaz et al. [2016](/article/10.1007/s00294-020-01057-z#ref-CR51 "Vaz B, Popovic M, Newman JA, Fielden J, Aitkenhead H, Halder S, Singh AN, Vendrell I, Fischer R, Torrecilla I, Drobnitzky N, Freire R, Amor DJ, Lockhart PJ, Kessler BM, McKenna GW, Gileadi O, Ramadan K (2016) Metalloprotease SPRTN/DVC1 orchestrates replication-coupled DNA-protein crosslink repair. Mol Cell 64:704–719. https://doi.org/10.1016/j.molcel.2016.09.032

")). To investigate how formaldehyde-induced DNA lesions are repaired, we first evaluated the involvement of S. pombe Wss1 homologs, Wss1 and Wss2 (Noguchi et al. [2017](/article/10.1007/s00294-020-01057-z#ref-CR36 "Noguchi C, Grothusen G, Anandarajan V, Martinez-Lage Garcia M, Terlecky D, Corzo K, Tanaka K, Nakagawa H, Noguchi E (2017) Genetic controls of DNA damage avoidance in response to acetaldehyde in fission yeast. Cell Cycle 16:45–58. https://doi.org/10.1080/15384101.2016.1237326

")), in formaldehyde tolerance. We found that _wss1_∆ and _wss2_∆ cells are mildly sensitive to formaldehyde (Fig. 4a, b). We also tested formaldehyde sensitivity of _wss1_∆ _wss2_∆ double mutant cells; _wss1_∆ _wss2_∆ cells were more sensitive to formaldehyde than either single mutant cells (Fig. 4b), suggesting that Wss1 and Wss2 have redundant functions in formaldehyde tolerance. To increase the burden of formaldehyde in Wss1- or Wss2-deficient cells, we constructed _wss1_∆ _fmd1_∆ and _wss2_∆ _fmd1_∆ double mutants. These double mutants displayed much stronger formaldehyde sensitivity than corresponding single mutants (Fig. 4a), suggesting the involvement of S. pombe Wss1/Wss2 proteases in the repair of formaldehyde-induced DPCs (Fig. 3a).

Fig. 4

Fig. 4

The alternative text for this image may have been generated using AI.

Full size image

DPC proteases are involved in formaldehyde tolerance. a, b Formaldehyde sensitivity of the indicated cells was evaluated as described in Fig. 1. _wss1_∆ and wss2 cells show mild sensitivity to formaldehyde. Formaldehyde sensitivity of these mutants further increased when combined with _fmd1_∆ or _swi1_∆. c Five-fold serial dilutions of the indicated cells were plated on YES agar medium supplemented with 0, 2 or 5 µM of camptothecin (CPT) and incubated for 3–4 days at 32 °C. d Formaldehyde-induced DPCs results in accumulation of DNA damage. Rad52-YFP-expressing cells of the indicated genotypes were treated with or without 10 mM acetaldehyde. Rad52-YFP foci analysis was performed as described in Fig. 2a. Student’s _t_-test, *p < 0.05, **p < 0.01

Because Wss1/Spartan/DVC1-mediated DPC removal appears to be dependent on DNA replication, Spartan-related proteins are proposed to function at the replication fork (Lopez-Mosqueda et al. 2016; Stingele et al. [2016](/article/10.1007/s00294-020-01057-z#ref-CR47 "Stingele J, Bellelli R, Alte F, Hewitt G, Sarek G, Maslen SL, Tsutakawa SE, Borg A, Kjaer S, Tainer JA, Skehel JM, Groll M, Boulton SJ (2016) Mechanism and regulation of DNA-protein crosslink repair by the DNA-dependent metalloprotease SPRTN. Mol Cell 64:688–703. https://doi.org/10.1016/j.molcel.2016.09.031

"); Vaz et al. [2016](/article/10.1007/s00294-020-01057-z#ref-CR51 "Vaz B, Popovic M, Newman JA, Fielden J, Aitkenhead H, Halder S, Singh AN, Vendrell I, Fischer R, Torrecilla I, Drobnitzky N, Freire R, Amor DJ, Lockhart PJ, Kessler BM, McKenna GW, Gileadi O, Ramadan K (2016) Metalloprotease SPRTN/DVC1 orchestrates replication-coupled DNA-protein crosslink repair. Mol Cell 64:704–719. https://doi.org/10.1016/j.molcel.2016.09.032

")). Therefore, we deleted the swi1+ gene (required for replication fork protection) in _wss1_∆ and _wss2_∆ cells. Importantly, _wss1_∆ _swi1_∆ and _wss2_∆ _swi1_∆ cells showed slight, but reproducibly higher sensitivity to formaldehyde than single mutant cells (Fig. 4b). We also tested camptothecin (CPT) sensitivity of these mutant cells because CPT traps topoisomerase onto DNA, forming stable DPCs (Pommier [2006](/article/10.1007/s00294-020-01057-z#ref-CR37 "Pommier Y (2006) Topoisomerase I inhibitors: camptothecins and beyond. Nat Rev Cancer 6:789–802. https://doi.org/10.1038/nrc1977

")). The major pathway that removes CPT-mediated topoisomerase-DNA cleavage complex involves the Tdp1 tyrosyl-DNA-phosphodiesterase (Pommier et al. [2006](/article/10.1007/s00294-020-01057-z#ref-CR38 "Pommier Y, Barcelo JM, Rao VA, Sordet O, Jobson AG, Thibaut L, Miao ZH, Seiler JA, Zhang H, Marchand C, Agama K, Nitiss JL, Redon C (2006) Repair of topoisomerase I-mediated DNA damage. Prog Nucleic Acid Res Mol Biol 81:179–229. https://doi.org/10.1016/s0079-6603(06)81005-6

")). In budding yeast, which has only one Wss1 homolog, _wss1_∆ cells failed to show CPT sensitivity unless the tdp1 gene is also deleted (Stingele et al. [2014](/article/10.1007/s00294-020-01057-z#ref-CR46 "Stingele J, Schwarz MS, Bloemeke N, Wolf PG, Jentsch S (2014) A DNA-dependent protease involved in DNA-protein crosslink repair. Cell 158:327–338. https://doi.org/10.1016/j.cell.2014.04.053

")). Consistently, in fission yeast, _wss1_∆, _wss2_∆, and _wss1_∆ _wss2_∆ cells were not significantly sensitive to CPT (Fig. 4c). However, _wss1_∆ _swi1_∆ and _wss2_∆ _swi1_∆ cells were more sensitive to CPT than single mutant cells (Fig. 4c). These results imply that DPC accumulated in _wss1_∆ or _wss2_∆ cells induce DNA replication problems that are alleviated by the replication fork protection protein Swi1.

To further strengthen the effect of DPCs on genomic integrity, we monitored Rad52-YFP foci in _wss1_∆ and _wss2_∆ cells treated with formaldehyde. As shown in Fig. 4d, these cells displayed significantly more formaldehyde-induced Rad52-YFP foci than wild-type cells. We also assessed formaldehyde-induced DNA damage in _wss1_∆ _wss2_∆ double mutant cells; there was no further accumulation of DNA damage in comparison to single mutant cells (Fig. 4d). Because the difference between _wss2_∆ and _wss1_∆ _wss2_∆ in formaldehyde sensitivity assay is small (Fig. 4b), it is possible that this small difference is not reflected in the Rad52 foci assay. Nevertheless, our results indicate that the DPC proteases Wss1 and Wss2 are involved in preventing DNA damage in response to formaldehyde.

HR and NER play critical roles in cellular tolerance to formaldehyde

It is suggested that DPCs generate bulky DNA lesions that require nucleotide excision repair (NER) and/or homologous recombination (HR) depending on the sizes of proteins or peptides crosslinked onto DNA (Kawanishi et al. 2014; Nakano et al. [2009](/article/10.1007/s00294-020-01057-z#ref-CR30 "Nakano T, Katafuchi A, Matsubara M, Terato H, Tsuboi T, Masuda T, Tatsumoto T, Pack SP, Makino K, Croteau DL, Van Houten B, Iijima K, Tauchi H, Ide H (2009) Homologous recombination but not nucleotide excision repair plays a pivotal role in tolerance of DNA-protein cross-links in mammalian cells. J Biol Chem 284:27065–27076. https://doi.org/10.1074/jbc.M109.019174

"), [2007](/article/10.1007/s00294-020-01057-z#ref-CR29 "Nakano T, Morishita S, Katafuchi A, Matsubara M, Horikawa Y, Terato H, Salem AM, Izumi S, Pack SP, Makino K, Ide H (2007) Nucleotide excision repair and homologous recombination systems commit differentially to the repair of DNA-protein crosslinks. Mol Cell 28:147–158. https://doi.org/10.1016/j.molcel.2007.07.029

")). Indeed, formaldehyde treatment resulted in nuclear accumulation of Rad52 DNA repair foci (Fig. 2a, b), and cells deficient for Rad51 or Rad52 recombinases are highly sensitive to formaldehyde (Fig. 2c). We next determined formaldehyde sensitivity of S. pombe cells that are deficient for the Swi10ERCC1-Swi9XPF/FANCQ nuclease complex required for NER. As shown in Fig. 5a, _swi9_∆ and _swi10_∆ cells were both highly sensitive to formaldehyde. Thus, HR and NER processes are critical for cellular tolerance to formaldehyde.

Fig. 5

Fig. 5

The alternative text for this image may have been generated using AI.

Full size image

NER and BER are involved in the repair of formaldehyde-induced DNA lesions. a Formaldehyde sensitivity of the indicated cells was evaluated as described in Fig. 1. BER (_nth1_∆ and _apn2_∆) and NER (_swi9_∆ and _swi10_∆) mutants were sensitive to formaldehyde. b, c Formaldehyde sensitivity assays of the indicated cells were performed as described in Fig. 1. For each condition, all strains were plated on the same plate for formaldehyde sensitivity assay. Loss of Rad3 (_rad3_∆) further sensitizes BER and NER mutants to formaldehyde. d Five-fold serial dilutions of the indicated cells were plated on YES agar medium supplemented with the indicated amounts of acetaldehyde. Cells were then incubated for 3–5 days in a sealed condition to prevent acetaldehyde evaporation. NER but not BER mutants were sensitive to acetaldehyde

When cells have DNA damage, cells need to activate Rad3ATR-mediated cell cycle checkpoints to allow time for efficient DNA repair (Hustedt et al. [2013](/article/10.1007/s00294-020-01057-z#ref-CR17 "Hustedt N, Gasser SM, Shimada K (2013) Replication checkpoint: tuning and coordination of replication forks in s phase. Genes 4:388–434. https://doi.org/10.3390/genes4030388

"); Noguchi 2010). Therefore, we constructed _swi9_∆ _rad3_∆ and _swi10_∆ _rad3_∆ double mutant cells. These cells showed much higher formaldehyde sensitivity than either corresponding single mutant cells (Fig. 5b). Thus, our results are consistent with the notion that formaldehyde-induced DNA lesions activate the Rad3ATR checkpoint kinase to allow time for DNA repair by NER.

The Fanconi anemia DNA repair pathway is involved in formaldehyde-induced stress response

Although formaldehyde is often used as a DPC-inducing agent, it causes other lesions including ICLs and base adducts (Kawanishi et al. 2014). The FA pathway is critical for the repair of ICLs and is known to coordinate NER, HR, and TLS process to remove ICLs (Fig. 1a) (Ceccaldi et al. [2016](/article/10.1007/s00294-020-01057-z#ref-CR7 "Ceccaldi R, Sarangi P, D'Andrea AD (2016) The Fanconi anaemia pathway: new players and new functions. Nat Rev Mol Cell Biol 17:337–349. https://doi.org/10.1038/nrm.2016.48

")). We found that S. pombe cells deficient for Swi9XPF/FANCQ (an NER factor, Fig. 5a) and Rad51FANCO (an HR factor, Fig. 2c) were highly sensitive to formaldehyde. Importantly, these factors are also involved in the FA pathway (Ceccaldi et al. [2016](/article/10.1007/s00294-020-01057-z#ref-CR7 "Ceccaldi R, Sarangi P, D'Andrea AD (2016) The Fanconi anaemia pathway: new players and new functions. Nat Rev Mol Cell Biol 17:337–349. https://doi.org/10.1038/nrm.2016.48

")). In addition, we determined formaldehyde sensitivity of cells deficient for other proteins involved in the FA pathway, including Fml1FANCM and Chl1FANCJ/ChlR1. _fml1_∆ and _chl1_∆ cells were sensitive to formaldehyde (Figs. 3b, 6a). In addition, _fml1_∆ _fmd1_∆ and _chl1_∆ _fmd1_∆ cells were much more sensitive to formaldehyde than corresponding single mutant cells (Fig. 3b). Furthermore, _rad3_∆ _fml1_∆ cells displayed stronger formaldehyde sensitivity than either single mutant cells (Fig. 6a). Thus, our results are consistent with the notion that formaldehyde induces ICLs.

Fig. 6

Fig. 6

The alternative text for this image may have been generated using AI.

Full size image

The Fanconi anemia pathway is involved in the repair of formaldehyde-induced DNA damage. Formaldehyde sensitivity of the indicated cells was evaluated as described in Fig. 1. a FA mutant show formaldehyde sensitivity. b HR and the FA pathway function in separate pathways required for formaldehyde tolerance. c NER functions independently of the FA pathway in response to formaldehyde. d TLS mutants show formaldehyde sensitivity. (e) Fan1FAN1 and Pso2SNM1 nucleases are involved in repair of formaldehyde-induced DNA damage

Studies demonstrated that ICLs interfere with the DNA replication process and that ICLs are repaired in a DNA replication-dependent manner (Ceccaldi et al. [2016](/article/10.1007/s00294-020-01057-z#ref-CR7 "Ceccaldi R, Sarangi P, D'Andrea AD (2016) The Fanconi anaemia pathway: new players and new functions. Nat Rev Mol Cell Biol 17:337–349. https://doi.org/10.1038/nrm.2016.48

")). For this reason, we tested the involvement of the replication fork protection protein Swi1Timeless in the repair of formaldehyde-induced ICLs. _swi1_∆ _chl1_∆ and _swi1_∆ _fml1_∆ cells were more sensitive to formaldehyde than corresponding single mutant cells (Fig. 6a). Considering that Chl1FANCJ/ChlR1 and Fml1FANCM function in the FA pathway responsible for ICL repair, our results suggest that formaldehyde-induced ICLs also hamper the DNA replication process.

Because it is generally accepted that the FA pathway coordinates NER and HR pathways at the replication fork (Fig. 1a), we evaluated genetic interaction among the fml1 (FA), rad52 (NER), and swi9 (NER) genes. Strikingly, _fml1_∆ _rad52_∆ double mutants were much more sensitive to formaldehyde than either single mutants (Fig. 6b). In addition, _fml1_∆ _swi9_∆ double mutants displayed higher formaldehyde sensitivity than _fml1_∆ or _swi9_∆ cells (Fig. 6c). Furthermore, _swi1_∆ _swi9_∆ and _swi1_∆ _rad52_∆ double mutants were more sensitive to formaldehyde than corresponding single mutants (Fig. 6b, c). These results suggest that, in response to formaldehyde-induced DNA lesions, Fml1FANCM functions independently of HR, NER, and the Swi1-mediated replication fork protection mechanism. Although HR and NER may also work downstream of the FA pathway as proposed for ICL repair, our results suggest that formaldehyde-induced DNA lesions also utilize HR- and NER-mediated repair mechanisms independently of the FA pathway.

We next investigated the role of translesion synthesis (TLS) in formaldehyde stress response because the FA pathway also coordinates TLS to fill the gap in the DNA strand generated during the ICL repair process. Loss of individual TLS polymerases Kpa1/DinB (Pol κ) or Rev3 (Pol ζ), but not Eso1 (Pol η), resulted in mild formaldehyde sensitivity (Fig. 6d). We also included _eso1_∆ _kpa1_∆ _rev3_∆ triple mutant cells (_TLS_∆), which are also sensitive to formaldehyde although they showed similar formaldehyde sensitivity to that of _rev3_∆ single mutant cells (Fig. 6d). These results suggest that TLS polymerases play a critical role in the repair of formaldehyde-induced DNA lesions.

BER is involved in the repair of formaldehyde-induced DNA lesions

Formaldehyde is known to react with deoxynucleotides, forming base adducts (monoadducts), which could be removed by the base excision repair (BER) pathway (Kawanishi et al. 2014). Accordingly, we determined formaldehyde sensitivity of BER mutants. These include cells defective in endonuclease III Nth1 (_nth1_∆) and AP-endonuclease Apn2 (_apn2_∆) (Sugimoto et al. [2005](/article/10.1007/s00294-020-01057-z#ref-CR48 "Sugimoto T, Igawa E, Tanihigashi H, Matsubara M, Ide H, Ikeda S (2005) Roles of base excision repair enzymes Nth1p and Apn2p from Schizosaccharomyces pombe in processing alkylation and oxidative DNA damage. DNA Repair 4:1270–1280. https://doi.org/10.1016/j.dnarep.2005.06.009

")). These cells show considerable sensitivity to formaldehyde, indicating that BER is involved in the repair of formaldehyde-induced DNA lesion (Fig. 5a). In addition, _nth1_∆ _rad3_∆ and _apn2_∆ _rad3_∆ cells were more sensitive to formaldehyde than corresponding single mutant cells (Fig. 5c).

Interestingly, NER mutants (_swi9_∆ and _swi10_∆) were more sensitive to formaldehyde than BER mutants (_nth1_∆ and _apn2_∆) (Fig. 5a), suggesting that bulky lesions generated by formaldehyde are more toxic than formaldehyde-induced base adducts, although base adducts appear to cause significant DNA damage in formaldehyde-treated cells. We also noted that BER mutants were not significantly sensitive to acetaldehyde, whereas NER mutants displayed hypersensitivity to acetaldehyde (Fig. 5d), suggesting that, compared to acetaldehyde, formaldehyde induces more toxic and/or significant amounts of base adducts (Fig. 7a, b). Taken together, our results are consistent with the notion that BER plays an important role in the repair of formaldehyde-induced DNA lesions.

Fig. 7

Fig. 7

The alternative text for this image may have been generated using AI.

Full size image

Models of aldehyde DNA damage response. a The major lesions induced by formaldehyde are DPCs that are repaired by NER and HR. Formaldehyde also induces base adducts and ICLs that are repaired by BER and the FA pathway, respectively. b Acetaldehyde causes DPCs and ICLs. Base adducts are not major acetaldehyde-induced DNA lesions. c Endonucleases involved in formaldehyde and aldehyde tolerance

Endonucleases involved in the repair of formaldehyde-induced DNA lesions

Endonucleases play a critical role in crosslink repair. These nucleases include Swi9XPF/FANCQ-Swi10ERCC1, Slx1-Slx4FANCP, Fan1FAN1 and Pso2SNM1 in S. pombe (Coulon et al. [2004](/article/10.1007/s00294-020-01057-z#ref-CR10 "Coulon S, Gaillard PHL, Chahwan C, McDonald WH, Yates JR 3rd, Russell P (2004) Slx1-Slx4 are subunits of a structure-specific endonuclease that maintains ribosomal DNA in fission yeast. Mol Biol Cell 15:71–80. https://doi.org/10.1091/mbc.e03-08-0586

"); Fontebasso et al. [2013](/article/10.1007/s00294-020-01057-z#ref-CR13 "Fontebasso Y, Etheridge TJ, Oliver AW, Murray JM, Carr AM (2013) The conserved Fanconi anemia nuclease Fan1 and the SUMO E3 ligase Pli1 act in two novel Pso2-independent pathways of DNA interstrand crosslink repair in yeast. DNA Repair 12:1011–1023. https://doi.org/10.1016/j.dnarep.2013.10.003

"); Schmidt et al. [1989](/article/10.1007/s00294-020-01057-z#ref-CR44 "Schmidt H, Kapitza-Fecke P, Stephen ER, Gutz H (1989) Some of the swi genes of Schizosaccharomyces pombe also have a function in the repair of radiation damage. Curr Genet 16:89–94. https://doi.org/10.1007/bf00393400

")). We previously demonstrated that Swi9XPF/FANCQ-Swi10ERCC1 is the major endonuclease that prevents acetaldehyde- and cisplatin-induced DNA damage in S. pombe (Noguchi et al. [2017](/article/10.1007/s00294-020-01057-z#ref-CR36 "Noguchi C, Grothusen G, Anandarajan V, Martinez-Lage Garcia M, Terlecky D, Corzo K, Tanaka K, Nakagawa H, Noguchi E (2017) Genetic controls of DNA damage avoidance in response to acetaldehyde in fission yeast. Cell Cycle 16:45–58. https://doi.org/10.1080/15384101.2016.1237326

")). In this previous study, we demonstrated that _slx4_∆, _fan1_∆, and _pso2_∆ cells failed to show significant sensitivity to acetaldehyde while _swi9_∆ and _swi10_∆ cells were sensitive. Similarly, among the nuclease mutants tested, only _swi9_∆ and _swi10_∆ cells were sensitive to cisplatin, an ICL-inducing agent (Noguchi et al. [2017](/article/10.1007/s00294-020-01057-z#ref-CR36 "Noguchi C, Grothusen G, Anandarajan V, Martinez-Lage Garcia M, Terlecky D, Corzo K, Tanaka K, Nakagawa H, Noguchi E (2017) Genetic controls of DNA damage avoidance in response to acetaldehyde in fission yeast. Cell Cycle 16:45–58. https://doi.org/10.1080/15384101.2016.1237326

")). Interestingly, we found that _fan1_∆ and _pso2_∆ cells showed considerable sensitivity to formaldehyde (Fig. 6d). As was the case with acetaldehyde (Noguchi et al. [2017](/article/10.1007/s00294-020-01057-z#ref-CR36 "Noguchi C, Grothusen G, Anandarajan V, Martinez-Lage Garcia M, Terlecky D, Corzo K, Tanaka K, Nakagawa H, Noguchi E (2017) Genetic controls of DNA damage avoidance in response to acetaldehyde in fission yeast. Cell Cycle 16:45–58. https://doi.org/10.1080/15384101.2016.1237326

")), _slx4_∆ cells were not sensitive to formaldehyde (data not shown), whereas _swi9_∆ and _swi10_∆ showed considerable sensitivity to formaldehyde (Fig. 5a). These data suggest that, in addition to Swi9XPF/FANCQ-Swi10ERCC1, Fan1FAN1 and Pso2SNM1 nucleases play critical roles in the removal of formaldehyde-induced DNA lesions (Fig. 7c). Thus, formaldehyde and acetaldehyde appear to cause different spectrums of DNA lesions.

Discussion

In this report, we dissected genetic mechanisms involved in cellular tolerance to formaldehyde. We have identified fission yeast genes involved in formaldehyde detoxification. We also evaluated formaldehyde sensitivity of various DNA repair mutants to understand the nature of formaldehyde-induced DNA lesions. Our studies suggest that formaldehyde induces a variety of DNA lesions, including DPCs, ICLs, and base adducts.

Fmd1 was found to be a major formaldehyde dehydrogenase in S. pombe. Importantly, _fmd1_∆ cells are highly sensitive to formaldehyde but not to acetaldehyde (Fig. 1). Although we did not examine the effect of other aldehydic substances, Fmd1 may function specifically to detoxify formaldehyde, whereas other dehydrogenase including Adh1 and Atd1 aid in the removal of both acetaldehyde and formaldehyde. Importantly, both aldehydic substances appear to impede the DNA replication process as loss of Swi1, a component of the replication fork protection complex, sensitizes S. pombe cells to acetaldehyde (Noguchi et al. [2017](/article/10.1007/s00294-020-01057-z#ref-CR36 "Noguchi C, Grothusen G, Anandarajan V, Martinez-Lage Garcia M, Terlecky D, Corzo K, Tanaka K, Nakagawa H, Noguchi E (2017) Genetic controls of DNA damage avoidance in response to acetaldehyde in fission yeast. Cell Cycle 16:45–58. https://doi.org/10.1080/15384101.2016.1237326

")) and formaldehyde (Fig. 3). Consistently, the Rad3ATR-Rad26ATRIP complex, which is activated in response to replication stress, is also required for cellular tolerance to acetaldehyde (Noguchi et al. [2017](/article/10.1007/s00294-020-01057-z#ref-CR36 "Noguchi C, Grothusen G, Anandarajan V, Martinez-Lage Garcia M, Terlecky D, Corzo K, Tanaka K, Nakagawa H, Noguchi E (2017) Genetic controls of DNA damage avoidance in response to acetaldehyde in fission yeast. Cell Cycle 16:45–58. https://doi.org/10.1080/15384101.2016.1237326

")) and formaldehyde (Fig. 3).

Cells must remove aldehyde-induced DNA adducts to resume the DNA replication process. The adduct removal process utilized by cells depends on the types of DNA adducts encountered. Among the DNA repair pathways we examined, HR and NER processes have the most significant roles in formaldehyde tolerance. This was similar to the effect of acetaldehyde on DNA repair mechanisms (Noguchi, et al. [2017](/article/10.1007/s00294-020-01057-z#ref-CR36 "Noguchi C, Grothusen G, Anandarajan V, Martinez-Lage Garcia M, Terlecky D, Corzo K, Tanaka K, Nakagawa H, Noguchi E (2017) Genetic controls of DNA damage avoidance in response to acetaldehyde in fission yeast. Cell Cycle 16:45–58. https://doi.org/10.1080/15384101.2016.1237326

")) (Fig. 7a, b). In addition, the FA pathway, which is involved in ICL repair, also plays an important role in DNA repair of both formaldehyde- and acetaldehyde-induced DNA lesions. The FA pathway may not be as critical as HR or NER because mutants in the FA pathway are only mildly sensitive to both formaldehyde and acetaldehyde, while HR and NER mutants show strong aldehyde sensitivity. Furthermore, our studies demonstrated that HR and NER function independently of the FA pathway in response to formaldehyde. Thus, although the FA pathway coordinates NER and HR processes in response to ICLs, ICLs may not represent the major DNA lesions generated by formaldehyde (Fig. 7a). Rather, formaldehyde-induced DNA lesions are largely repaired by NER and HR in a manner independent of the FA pathway (Fig. 7a).

As previously suggested, DPCs may represent the major DNA lesions induced by formaldehyde. Indeed, cells deficient for Wss1/2 DPC proteases showed sensitivity to formaldehyde. Furthermore, such DPCs appear to cause replication stress because Swi1 (a fork protection complex subunit) and Rad3ATR-Rad26ATRIP (central for the DNA replication stress checkpoint (Hustedt et al. [2013](/article/10.1007/s00294-020-01057-z#ref-CR17 "Hustedt N, Gasser SM, Shimada K (2013) Replication checkpoint: tuning and coordination of replication forks in s phase. Genes 4:388–434. https://doi.org/10.3390/genes4030388

"), Noguchi 2010)) are also required for cellular tolerance to formaldehyde (Fig. 3). Studies demonstrated that DPCs are repaired by NER and HR depending on the sizes of crosslinked proteins (Nakano et al. [2009](/article/10.1007/s00294-020-01057-z#ref-CR30 "Nakano T, Katafuchi A, Matsubara M, Terato H, Tsuboi T, Masuda T, Tatsumoto T, Pack SP, Makino K, Croteau DL, Van Houten B, Iijima K, Tauchi H, Ide H (2009) Homologous recombination but not nucleotide excision repair plays a pivotal role in tolerance of DNA-protein cross-links in mammalian cells. J Biol Chem 284:27065–27076. https://doi.org/10.1074/jbc.M109.019174

"), [2007](/article/10.1007/s00294-020-01057-z#ref-CR29 "Nakano T, Morishita S, Katafuchi A, Matsubara M, Horikawa Y, Terato H, Salem AM, Izumi S, Pack SP, Makino K, Ide H (2007) Nucleotide excision repair and homologous recombination systems commit differentially to the repair of DNA-protein crosslinks. Mol Cell 28:147–158. https://doi.org/10.1016/j.molcel.2007.07.029

")). It appears that NER removes DPCs only when the crosslinked proteins are smaller than 8–10 kDa (Nakano et al. [2009](/article/10.1007/s00294-020-01057-z#ref-CR30 "Nakano T, Katafuchi A, Matsubara M, Terato H, Tsuboi T, Masuda T, Tatsumoto T, Pack SP, Makino K, Croteau DL, Van Houten B, Iijima K, Tauchi H, Ide H (2009) Homologous recombination but not nucleotide excision repair plays a pivotal role in tolerance of DNA-protein cross-links in mammalian cells. J Biol Chem 284:27065–27076. https://doi.org/10.1074/jbc.M109.019174

")). Considering that most proteins are much larger than these sizes, HR appears to play a critical role in DPC removal. However, Wss1-related proteases reduce protein sizes to promote NER-mediated DPC repair. Such a mechanism seems to be very important in cellular tolerance to formaldehyde, as NER mutants were highly sensitive to formaldehyde (Fig. 5).

Interestingly, our investigation revealed that formaldehyde-induced DNA lesions require BER to be repaired, whereas BER is dispensable for the repair of acetaldehyde-induced lesions (Fig. 5a, d). Such results strongly indicate that base adducts also make up a major portion of formaldehyde-induced DNA lesions and that there are fundamental differences in types and ratios of formaldehyde- and acetaldehyde-induced DNA adducts (Fig. 7a, b). In addition to a variety of formaldehyde-induced base adducts, which themselves cause genomic instability, once formaldehyde reacts with nucleotides that contain amino group, methylene bridges can be formed, resulting in the formation of DPCs (Lu et al. [2010](/article/10.1007/s00294-020-01057-z#ref-CR25 "Lu K, Ye W, Zhou L, Collins LB, Chen X, Gold A, Ball LM, Swenberg JA (2010) Structural characterization of formaldehyde-induced cross-links between amino acids and deoxynucleosides and their oligomers. J Am Chem Soc 132:3388–3399. https://doi.org/10.1021/ja908282f

")). DPCs are thought to be the chief genotoxic effects following exposure to formaldehyde, although formaldehyde can also induce ICLs between adjacent guanine nucleotides (Lu et al. [2010](/article/10.1007/s00294-020-01057-z#ref-CR25 "Lu K, Ye W, Zhou L, Collins LB, Chen X, Gold A, Ball LM, Swenberg JA (2010) Structural characterization of formaldehyde-induced cross-links between amino acids and deoxynucleosides and their oligomers. J Am Chem Soc 132:3388–3399. https://doi.org/10.1021/ja908282f

")). Consistently, our results suggest that DPC repair processes, including NER, have critical roles in formaldehyde tolerance (Fig. 7a). Another aldehydic agent, acetaldehyde, also forms base adducts including _N_2-ethyl-2′-deoxyguanosine, although this adducts may not have a significant impact on genomic integrity (Brooks and Zakhari [2014](/article/10.1007/s00294-020-01057-z#ref-CR5 "Brooks PJ, Zakhari S (2014) Acetaldehyde and the genome: beyond nuclear DNA adducts and carcinogenesis. Environ Mol Mutagen 55:77–91. https://doi.org/10.1002/em.21824

")). However, another major acealdehyde-induce DNA adducts, 1,_N_2-propano-2′-deoxyguanosine allows for the formation of DPCs and ICLs and hampers the DNA replication process, leading to genomic instability (Brooks and Zakhari [2014](/article/10.1007/s00294-020-01057-z#ref-CR5 "Brooks PJ, Zakhari S (2014) Acetaldehyde and the genome: beyond nuclear DNA adducts and carcinogenesis. Environ Mol Mutagen 55:77–91. https://doi.org/10.1002/em.21824

")). Consistently, BER appears to be less important for acetaldehyde response, whereas mutations the NER and the FA pathways cause significant sensitivity to acetaldehyde (Fig. 7b) (Noguchi et al. [2017](/article/10.1007/s00294-020-01057-z#ref-CR36 "Noguchi C, Grothusen G, Anandarajan V, Martinez-Lage Garcia M, Terlecky D, Corzo K, Tanaka K, Nakagawa H, Noguchi E (2017) Genetic controls of DNA damage avoidance in response to acetaldehyde in fission yeast. Cell Cycle 16:45–58. https://doi.org/10.1080/15384101.2016.1237326

")).

We also found the involvement of different endonucleases in the repair of formaldehyde- and acetaldehyde-induced DNA damage. We previously found that Swi9XPF/FANCQ-Swi10ERCC1 plays the major role in the removal of acetaldehyde-induced DNA lesions. This was also the case for DNA repair in response to cisplatin, an ICL-inducing agent. It is highly possible that both DPC- and ICL-repair utilize the Swi9XPF/FANCQ-Swi10ERCC1 nuclease. Interestingly, Fan1FAN1 and Pso2SNM1 appear to be dispensable for DNA repair in response to acetaldehyde and cisplatin (Noguchi et al. [2017](/article/10.1007/s00294-020-01057-z#ref-CR36 "Noguchi C, Grothusen G, Anandarajan V, Martinez-Lage Garcia M, Terlecky D, Corzo K, Tanaka K, Nakagawa H, Noguchi E (2017) Genetic controls of DNA damage avoidance in response to acetaldehyde in fission yeast. Cell Cycle 16:45–58. https://doi.org/10.1080/15384101.2016.1237326

")). However, in addition to Swi9XPF/FANCQ-Swi10ERCC1, Fan1FAN1 and Pso2SNM1 are also critical for formaldehyde response (Fig. 7c). Further investigations are warranted to investigate the role of Fan1FAN1 and Pso2SNM1 nucleases in the removal of formaldehyde-induced DNA adducts.

In summary, our studies have provided a genetic basis of formaldehyde-induced DNA damage response in fission yeast. The formaldehyde concentrations used in our fission yeast-based experiments are much higher compared to possible human exposure levels to these carcinogens. However, considering that DNA repair mechanisms are highly similar between S. pombe and humans, our studies will help to build future studies to understand formaldehyde-induced genotoxicity and its effect on human health.

Abbreviations

BER:

Base excision repair

DPC:

DNA–protein crosslink

FA:

Fanconi anemia

FPC:

Fork protection complex

HR:

Homologous recombination

ICL:

Interstrand crosslink

NER:

Nucleotide excision repair

TLS:

Translesion synthesis

References

Download references

Acknowledgement

We thank Drs. Amanda Bird, Shogo Ikeda, Matthew O’Connel, Matthew Whitby, and National BioResource Project Japan for S. pombe strains. We also thank Sofia Acchione for technical assistance. The members of the Noguchi laboratory are thanked for their support and encouragement. This work was supported by Drexel University College of Medicine and the Aging Initiative at Drexel University College of Medicine (to E.N.).

Author information

Author notes

  1. Grant Grothusen
    Present address: School of Medicine, Cell and Molecular Biology Graduate Program, University of Pennsylvania, Philadelphia, PA, USA
  2. Daniel Terlecky
    Present address: West Chester University of Pennsylvania, Environmental Health Graduate Program, Philadelphia, PA, USA
  3. Vinesh Anandarajan, Chiaki Noguchi authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, 245 N. 15th Street, Philadelphia, PA, 19102, USA
    Vinesh Anandarajan, Chiaki Noguchi, Julia Oleksak, Grant Grothusen, Daniel Terlecky & Eishi Noguchi

Authors

  1. Vinesh Anandarajan
  2. Chiaki Noguchi
  3. Julia Oleksak
  4. Grant Grothusen
  5. Daniel Terlecky
  6. Eishi Noguchi

Corresponding author

Correspondence toEishi Noguchi.

Additional information

Communicated by M. Kupiec.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Rights and permissions

About this article

Cite this article

Anandarajan, V., Noguchi, C., Oleksak, J. et al. Genetic investigation of formaldehyde-induced DNA damage response in Schizosaccharomyces pombe.Curr Genet 66, 593–605 (2020). https://doi.org/10.1007/s00294-020-01057-z

Download citation

Keywords