title: Next-generation sequencing reveals the biological significance of the N(2),3-ethenoguanine lesion in vivo.
abstract: Etheno DNA adducts are a prevalent type of DNA damage caused by vinyl chloride (VC) exposure and oxidative stress. Etheno adducts are mutagenic and may contribute to the initiation of several pathologies; thus, elucidating the pathways by which they induce cellular transformation is critical. Although N(2),3-ethenoguanine (N(2),3-εG) is the most abundant etheno adduct, its biological consequences have not been well characterized in cells due to its labile glycosidic bond. Here, a stabilized 2'-fluoro-2'-deoxyribose analog of N(2),3-εG was used to quantify directly its genotoxicity and mutagenicity. A multiplex method involving next-generation sequencing enabled a large-scale in vivo analysis, in which both N(2),3-εG and its isomer 1,N(2)-ethenoguanine (1,N(2)-εG) were evaluated in various repair and replication backgrounds. We found that N(2),3-εG potently induces G to A transitions, the same mutation previously observed in VC-associated tumors. By contrast, 1,N(2)-εG induces various substitutions and frameshifts. We also found that N(2),3-εG is the only etheno lesion that cannot be repaired by AlkB, which partially explains its persistence. Both εG lesions are strong replication blocks and DinB, a translesion polymerase, facilitates the mutagenic bypass of both lesions. Collectively, our results indicate that N(2),3-εG is a biologically important lesion and may have a functional role in VC-induced or inflammation-driven carcinogenesis.
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, United States Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, United States.
authors.forename: Shiou-chi
authors.initials: SC
authors.lastname: Chang
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, United States Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, United States Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, United States.
authors.forename: Bogdan I
authors.initials: BI
authors.lastname: Fedeles
authors.affiliation: BioMicro Center, Massachusetts Institute of Technology, Cambridge, MA 02139, United States.
authors.forename: Jie
authors.initials: J
authors.lastname: Wu
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, United States Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, United States Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, United States.
authors.forename: James C
authors.initials: JC
authors.lastname: Delaney
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, United States Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, United States Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, United States.
authors.forename: Deyu
authors.initials: D
authors.lastname: Li
authors.affiliation: Department of Biochemistry, Vanderbilt University, Nashville, TN 37232, United States Center in Molecular Toxicology, Vanderbilt University, Nashville, TN 37232, United States.
authors.forename: Linlin
authors.initials: L
authors.lastname: Zhao
authors.affiliation: Department of Biochemistry, Vanderbilt University, Nashville, TN 37232, United States Center in Molecular Toxicology, Vanderbilt University, Nashville, TN 37232, United States Department of Chemistry, Vanderbilt University, Nashville, TN 37232, United States.
authors.forename: Plamen P
authors.initials: PP
authors.lastname: Christov
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, United States Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, United States.
authors.forename: Emily
authors.initials: E
authors.lastname: Yau
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, United States Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, United States Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, United States.
authors.forename: Vipender
authors.initials: V
authors.lastname: Singh
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, United States.
authors.forename: Marco
authors.initials: M
authors.lastname: Jost
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, United States Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, United States Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, United States.
authors.forename: Catherine L
authors.initials: CL
authors.lastname: Drennan
authors.affiliation: Department of Biochemistry, Vanderbilt University, Nashville, TN 37232, United States Center in Molecular Toxicology, Vanderbilt University, Nashville, TN 37232, United States Department of Chemistry, Vanderbilt University, Nashville, TN 37232, United States Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, TN 37232, United States.
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Marnett
authors.affiliation: Department of Biochemistry, Vanderbilt University, Nashville, TN 37232, United States Center in Molecular Toxicology, Vanderbilt University, Nashville, TN 37232, United States Department of Chemistry, Vanderbilt University, Nashville, TN 37232, United States Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, TN 37232, United States.
authors.forename: Carmelo J
authors.initials: CJ
authors.lastname: Rizzo
authors.affiliation: BioMicro Center, Massachusetts Institute of Technology, Cambridge, MA 02139, United States.
authors.forename: Stuart S
authors.initials: SS
authors.lastname: Levine
authors.affiliation: Department of Biochemistry, Vanderbilt University, Nashville, TN 37232, United States Center in Molecular Toxicology, Vanderbilt University, Nashville, TN 37232, United States Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, TN 37232, United States.
authors.forename: F Peter
authors.initials: FP
authors.lastname: Guengerich
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, United States Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, United States Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, United States jessig@mit.edu.
authors.forename: John M
authors.initials: JM
authors.lastname: Essigmann
mesh_terms: D018736:DNA Adducts / Q000737:chemistry; D004249:DNA Damage; D019951:DNA Polymerase beta / Q000378:metabolism; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000378:metabolism; D049308:Dioxygenases / Q000378:metabolism; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry; D059014:High-Throughput Nucleotide Sequencing; D016296:Mutagenesis; D009154:Mutation; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion
chemical_list: D018736:DNA Adducts; C015196:N(2),3-ethenoguanine; D006147:Guanine; D049308:Dioxygenases; D019951:DNA Polymerase beta; D045643:DNA Repair Enzymes
references.citation: Trends Microbiol. 2007 Feb;15(2):70-7
references.pmid: 17207624
references.citation: Cancer Epidemiol Biomarkers Prev. 2003 Nov;12(11 Pt 1):1241-7
references.pmid: 14652288
references.citation: Chem Res Toxicol. 2010 Sep 20;23(9):1485-91
references.pmid: 20799743
references.citation: Pediatr Dermatol. 2005 Nov-Dec;22(6):501-12
references.pmid: 16354251
references.citation: Nature. 1987 Nov 12-18;330(6144):186-8
references.pmid: 3313061
references.citation: Bioinformatics. 2009 Aug 15;25(16):2078-9
references.pmid: 19505943
references.citation: Microbiol Mol Biol Rev. 2009 Mar;73(1):134-54
references.pmid: 19258535
references.citation: Nat Protoc. 2007;2(11):2728-33
references.pmid: 18007608
references.citation: PLoS One. 2014 Apr 14;9(4):e94716
references.pmid: 24733044
references.citation: Angew Chem Int Ed Engl. 2012 May 29;51(22):5466-9
references.pmid: 22488769
references.citation: PLoS One. 2013 Sep 16;8(9):e72993
references.pmid: 24066027
references.citation: Neuro Oncol. 2010 Jul;12(7):679-86
references.pmid: 20164241
references.citation: Proc Natl Acad Sci U S A. 1975 Aug;72(8):3190-3
references.pmid: 1059105
references.citation: Toxicol Sci. 2011 Mar;120 Suppl 1:S130-45
references.pmid: 21163908
references.citation: Cell. 2011 Mar 4;144(5):646-74
references.pmid: 21376230
references.citation: Carcinogenesis. 1995 Mar;16(3):613-7
references.pmid: 7697821
references.citation: Biochemistry. 2010 Mar 30;49(12):2615-26
references.pmid: 20201499
references.citation: Proc Natl Acad Sci U S A. 2007 Jan 16;104(3):755-60
references.pmid: 17213319
references.citation: IARC Monogr Eval Carcinog Risks Hum. 1991;52:1-544
references.pmid: 1683674
references.citation: Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):9974-8
references.pmid: 1946466
references.citation: Nat Rev Genet. 2010 Jan;11(1):31-46
references.pmid: 19997069
references.citation: Cancer Res. 1975 Nov;35(11 Pt. 2):3499-502
references.pmid: 1104156
references.citation: Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):905-21
references.pmid: 9757107
references.citation: Bioinformatics. 2014 Aug 1;30(15):2114-20
references.pmid: 24695404
references.citation: Science. 2013 Mar 29;339(6127):1546-58
references.pmid: 23539594
references.citation: Annu Rev Genomics Hum Genet. 2008;9:387-402
references.pmid: 18576944
references.citation: Cancer Res. 2004 Aug 15;64(16):5597-607
references.pmid: 15313897
references.citation: Methods Enzymol. 2006;408:1-15
references.pmid: 16793359
references.citation: Mutat Res. 1985 Nov-Dec;152(2-3):147-56
references.pmid: 3906388
references.citation: Cancer Res. 1990 Feb 1;50(3):480-2
references.pmid: 2404571
references.citation: Nature. 2010 Nov 11;468(7321):330-3
references.pmid: 21068844
references.citation: Biochemistry. 1997 May 20;36(20):6069-79
references.pmid: 9166777
references.citation: Cancer Biomark. 2005;1(1):5-14
references.pmid: 17192028
references.citation: J Biol Chem. 2005 Aug 19;280(33):29750-64
references.pmid: 15965231
references.citation: Chem Res Toxicol. 2000 Dec;13(12):1243-50
references.pmid: 11123965
references.citation: Biochemistry. 1998 Apr 14;37(15):5184-93
references.pmid: 9548749
references.citation: Toxicol Lett. 2002 Aug 5;134(1-3):71-7
references.pmid: 12191863
references.citation: Chem Res Toxicol. 2007 Nov;20(11):1601-11
references.pmid: 17941687
references.citation: Biochemistry. 1997 Sep 30;36(39):11933-43
references.pmid: 9305987
references.citation: Nat Methods. 2012 Mar 04;9(4):357-9
references.pmid: 22388286
references.citation: Bioinformatics. 2014 Mar 1;30(5):614-20
references.pmid: 24142950
references.citation: Mol Carcinog. 1991;4(6):450-4
references.pmid: 1793483
references.citation: Nucleic Acids Res. 1987 Aug 11;15(15):5963-71
references.pmid: 3627975
references.citation: N Engl J Med. 1988 Sep 1;319(9):525-32
references.pmid: 2841597
references.citation: Acc Chem Res. 2009 Apr 21;42(4):519-29
references.pmid: 19852088
references.citation: Carcinogenesis. 2000 Mar;21(3):361-70
references.pmid: 10688856
references.citation: Cancer Res. 2002 Sep 15;62(18):5189-95
references.pmid: 12234983
references.citation: Carcinogenesis. 1987 May;8(5):745-7
references.pmid: 3581434
references.citation: Carcinogenesis. 2001 Apr;22(4):535-45
references.pmid: 11285186
references.citation: Am J Public Health Nations Health. 1954 Feb;44(2):171-81
references.pmid: 13114492
references.citation: Nature. 2006 Jan 12;439(7073):225-8
references.pmid: 16407906
references.citation: Free Radic Biol Med. 2007 Oct 15;43(8):1109-20
references.pmid: 17854706
references.citation: Proc Natl Acad Sci U S A. 1994 Feb 1;91(3):1024-8
references.pmid: 8302827
references.citation: Chem Res Toxicol. 2005 Nov;18(11):1701-14
references.pmid: 16300379
references.citation: Nat Struct Mol Biol. 2005 Oct;12(10):855-60
references.pmid: 16200073
references.citation: Ann Ist Super Sanita. 2006;42(2):163-9
references.pmid: 17033136
references.citation: Cancer Res. 2001 Jul 15;61(14):5366-9
references.pmid: 11454676
references.citation: Chem Res Toxicol. 1995 Mar;8(2):278-83
references.pmid: 7766812
references.citation: Chem Res Toxicol. 2005 Apr;18(4):780-6
references.pmid: 15833039
references.citation: Nucleic Acids Res. 2011 Aug;39(14):5945-54
references.pmid: 21470959
references.citation: DNA Repair (Amst). 2004 Mar 4;3(3):257-65
references.pmid: 15177041
references.citation: Chem Res Toxicol. 1989 Jul-Aug;2(4):230-3
references.pmid: 2519779
references.citation: Anticancer Res. 2009 Jul;29(7):2727-37
references.pmid: 19596953
references.citation: Annu Rev Biochem. 1987;56:779-827
references.pmid: 3304147
references.citation: J Biol Chem. 2012 Oct 12;287(42):35516-35526
references.pmid: 22910910
references.citation: Proc Natl Acad Sci U S A. 2004 Sep 28;101(39):14051-6
references.pmid: 15381779
references.citation: Int J Oncol. 2006 Oct;29(4):957-64
references.pmid: 16964391
references.citation: J Am Chem Soc. 2014 Oct 1;136(39):13895-901
references.pmid: 25203306
references.citation: Carcinogenesis. 1996 Oct;17(10):2105-11
references.pmid: 8895475
references.citation: DNA Repair (Amst). 2005 May 2;4(5):583-93
references.pmid: 15811630
references.citation: Proc Natl Acad Sci U S A. 2008 Jun 24;105(25):8679-84
references.pmid: 18562283
references.citation: Front Biosci (Landmark Ed). 2011 Jun 01;16(8):3164-82
references.pmid: 21622227
references.citation: Carcinogenesis. 2010 Jan;31(1):59-70
references.pmid: 19875697
references.citation: Environ Mol Mutagen. 2002;40(1):36-40
references.pmid: 12211074