DNA of living cells is always exposed to damaging factors. To counteract the consequences of DNA lesions, cells have evolved several DNA repair systems, among which base excision repair is one of the most important systems. Many currently used antitumor drugs act by damaging DNA, and DNA repair often interferes with chemotherapy and radiotherapy in cancer cells. Tumors are usually extremely genetically heterogeneous, often bearing mutations in DNA repair genes. Thus, knowledge of the functionality of cancer-related variants of proteins involved in DNA damage response and repair is of great interest for personalization of cancer therapy. Although computational methods to predict the variant functionality have attracted much attention, at present, they are mostly based on sequence conservation and make little use of modern capabilities in computational analysis of 3D protein structures. We have used molecular dynamics (MD) to model the structures of 20 clinically observed variants of a DNA repair enzyme, 8-oxoguanine DNA glycosylase. In parallel, we have experimentally characterized the activity, thermostability, and DNA binding in a subset of these mutant proteins. Among the analyzed variants of 8-oxoguanine DNA glycosylase, three (I145M, G202C, and V267M) were significantly functionally impaired and were successfully predicted by MD. Alone or in combination with sequence-based methods, MD may be an important functional prediction tool for cancer-related protein variants of unknown significance. DNA of living cells is always exposed to damaging factors. To counteract the consequences of DNA lesions, cells have evolved several DNA repair systems, among which base excision repair is one of the most important systems. Many currently used antitumor drugs act by damaging DNA, and DNA repair often interferes with chemotherapy and radiotherapy in cancer cells. Tumors are usually extremely genetically heterogeneous, often bearing mutations in DNA repair genes. Thus, knowledge of the functionality of cancer-related variants of proteins involved in DNA damage response and repair is of great interest for personalization of cancer therapy. Although computational methods to predict the variant functionality have attracted much attention, at present, they are mostly based on sequence conservation and make little use of modern capabilities in computational analysis of 3D protein structures. We have used molecular dynamics (MD) to model the structures of 20 clinically observed variants of a DNA repair enzyme, 8-oxoguanine DNA glycosylase. In parallel, we have experimentally characterized the activity, thermostability, and DNA binding in a subset of these mutant proteins. Among the analyzed variants of 8-oxoguanine DNA glycosylase, three (I145M, G202C, and V267M) were significantly functionally impaired and were successfully predicted by MD. Alone or in combination with sequence-based methods, MD may be an important functional prediction tool for cancer-related protein variants of unknown significance. DNA of living cells is always exposed to a number of damaging factors, both endogenous and environmental. This causes modification of nucleobases and formation of abasic (apurinic/apyrimidinic, or AP, sites) and DNA breaks. The accumulated DNA lesions negatively affect the genome stability, leading to gene inactivation or changes in the properties of the encoded proteins. Most DNA lesions, such as deaminated, oxidized, and alkylated bases and single-strand breaks, are fixed by the base excision repair (BER) system (1Friedberg E.C. Walker G.C. Siede W. Wood R.D. Schultz R.A. Ellenberger T. DNA Repair and Mutagenesis. ASM Press, Washington, DC2006Google Scholar, 2Zharkov D.O. Base excision DNA repair.Cell Mol. Life Sci. 2008; 65: 1544-1565Crossref PubMed Scopus (201) Google Scholar). BER is initiated by the recognition of a lesion by one of the enzymes belonging to the group of DNA glycosylases, which hydrolyze the N-glycosidic bond between the nucleobase and C1′ of deoxyribose, forming an AP site. Another enzyme, AP endonuclease, then hydrolyzes the phosphodiester bond 5′ to the AP site. The repair cycle is finished after the incorporation of the missing deoxynucleoside monophosphate and ligation of the remaining nick (1Friedberg E.C. Walker G.C. Siede W. Wood R.D. Schultz R.A. Ellenberger T. DNA Repair and Mutagenesis. ASM Press, Washington, DC2006Google Scholar, 2Zharkov D.O. Base excision DNA repair.Cell Mol. Life Sci. 2008; 65: 1544-1565Crossref PubMed Scopus (201) Google Scholar). In addition to BER, several other DNA repair systems have been described in human cells: direct reversal, nucleotide excision repair, mismatch repair, recombination repair, and so forth (1Friedberg E.C. Walker G.C. Siede W. Wood R.D. Schultz R.A. Ellenberger T. DNA Repair and Mutagenesis. ASM Press, Washington, DC2006Google Scholar). Many currently used antitumor drugs act by damaging DNA, causing cytotoxicity in rapidly dividing cells (3Lawley P.D. Phillips D.H. DNA adducts from chemotherapeutic agents.Mutat. Res. 1996; 355: 13-40Crossref PubMed Scopus (217) Google Scholar). Thus, DNA repair systems, being absolutely required for genome maintenance in normal cells, often interfere with chemotherapy and radiotherapy in cancer cells. Inhibition of DNA repair is presently considered an efficient strategy to sensitize tumors toward drugs and ionizing radiation, and recently, inhibitors of poly(ADP-ribose)polymerase, a key BER regulator (4Fong P.C. Boss D.S. Yap T.A. Tutt A. Wu P. Mergui-Roelvink M. Mortimer P. Swaisland H. Lau A. O'Connor M.J. Ashworth A. Carmichael J. Kaye S.B. Schellens J.H.M. de Bono J.S. Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutation carriers.N. Engl. J. Med. 2009; 361: 123-134Crossref PubMed Scopus (2699) Google Scholar, 5Curtin N.J. Szabo C. Therapeutic applications of PARP inhibitors: anticancer therapy and beyond.Mol. Aspects Med. 2013; 34: 1217-1256Crossref PubMed Scopus (252) Google Scholar), as well as inhibitors of O6-methylguanine methyltransferase, an enzyme that repairs alkylated guanines (6Kaina B. Margison G.P. Christmann M. Targeting O6-methylguanine-DNA methyltransferase with specific inhibitors as a strategy in cancer therapy.Cell Mol. Life Sci. 2010; 67: 3663-3681Crossref PubMed Scopus (103) Google Scholar, 7Blumenthal D.T. Rankin C. Stelzer K.J. Spence A.M. Sloan A.E. Moore Jr., D.F. Padula G.D.A. Schulman S.B. Wade M.L. Rushing E.J. A phase III study of radiation therapy (RT) and O6-benzylguanine + BCNU versus RT and BCNU alone and methylation status in newly diagnosed glioblastoma and gliosarcoma: Southwest Oncology Group (SWOG) study S0001.Int. J. Clin. Oncol. 2015; 20: 650-658Crossref PubMed Scopus (32) Google Scholar), have been approved for clinical use. Several inhibitors of other targets in BER and other repair systems are presently at various stages of clinical trials. Tumors are usually extremely genetically heterogeneous because of their inherent genetic instability and fast clonal proliferation (8Burrell R.A. McGranahan N. Bartek J. Swanton C. The causes and consequences of genetic heterogeneity in cancer evolution.Nature. 2013; 501: 338-345Crossref PubMed Scopus (1248) Google Scholar, 9Beckman R.A. Loeb L.A. Evolutionary dynamics and significance of multiple subclonal mutations in cancer.DNA Repair. 2017; 56: 7-15Crossref PubMed Scopus (11) Google Scholar). Among other genetic changes, cancer cells often bear mutations in DNA repair genes (10Chae Y.K. Anker J.F. Carneiro B.A. Chandra S. Kaplan J. Kalyan A. Santa-Maria C.A. Platanias L.C. Giles F.J. Genomic landscape of DNA repair genes in cancer.Oncotarget. 2016; 7: 23312-23321Crossref PubMed Scopus (82) Google Scholar, 11Germano G. Lamba S. Rospo G. Barault L. Magrì A. Maione F. Russo M. Crisafulli G. Bartolini A. Lerda G. Siravegna G. Mussolin B. Frapolli R. Montone M. Morano F. et al.Inactivation of DNA repair triggers neoantigen generation and impairs tumour growth.Nature. 2017; 552: 116-120Crossref PubMed Scopus (239) Google Scholar). Most mutations in the protein-coding regions of cancer cell genomes are missense variants of unknown significance (12Kamps R. Brandão R.D. van den Bosch B.J. Paulussen A.D.C. Xanthoulea S. Blok M.J. Romano A. Next-generation sequencing in oncology: genetic diagnosis, risk prediction and cancer classification.Int. J. Mol. Sci. 2017; 18: 308Crossref Scopus (190) Google Scholar). To expand options for personalized cancer treatment, information about the functionality of variants of proteins involved in DNA damage response and repair is of great interest. Because at present it is easier to obtain genotype data than to functionally characterize variants of unknown significance, computational methods to predict the variant functionality have attracted much attention (13Castellana S. Mazza T. Congruency in the prediction of pathogenic missense mutations: state-of-the-art web-based tools.Brief Bioinform. 2013; 14: 448-459Crossref PubMed Scopus (64) Google Scholar, 14Khafizov K. Ivanov M.V. Glazova O.V. Kovalenko S.P. Computational approaches to study the effects of small genomic variations.J. Mol. Model. 2015; 21: 251Crossref PubMed Scopus (11) Google Scholar). However, direct benchmarking of such predictions against phenotypically characterized variants of cancer-related genes with known clinical significance still produces widely divergent results, preventing the use of currently used algorithms in practical cancer treatment personalization (15Martelotto L.G. Ng C.K.Y. De Filippo M.R. Zhang Y. Piscuoglio S. Lim R.S. Shen R. Norton L. Reis-Filho J.S. Weigelt B. Benchmarking mutation effect prediction algorithms using functionally validated cancer-related missense mutations.Genome Biol. 2014; 15: 484Crossref PubMed Scopus (85) Google Scholar). The development of new computational ways to predict the effect of mutations in protein-coding regions is therefore of utmost importance. At present, the majority of algorithms used for the prediction of the effects of mutations on the protein functions are based on the analysis of evolutionary conservation, changes in the amino acid physicochemical properties, and the available static structures rather than on the modeling of the dynamic behavior of the protein molecule (16Ng P.C. Henikoff S. Predicting the effects of amino acid substitutions on protein function.Annu. Rev. Genomics Hum. Genet. 2006; 7: 61-80Crossref PubMed Scopus (682) Google Scholar, 17Carter H. Chen S. Isik L. Tyekucheva S. Velculescu V.E. Kinzler K.W. Vogelstein B. Karchin R. Cancer-specific high-throughput annotation of somatic mutations: computational prediction of driver missense mutations.Cancer Res. 2009; 69: 6660-6667Crossref PubMed Scopus (310) Google Scholar, 18Kumar P. Henikoff S. Ng P.C. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm.Nat. Protoc. 2009; 4: 1073-1081Crossref PubMed Scopus (4557) Google Scholar, 19Adzhubei I.A. Schmidt S. Peshkin L. Ramensky V.E. Gerasimova A. Bork P. Kondrashov A.S. Sunyaev S.R. A method and server for predicting damaging missense mutations.Nat. Methods. 2010; 7: 248-249Crossref PubMed Scopus (8180) Google Scholar, 20González-Pérez A. López-Bigas N. Improving the assessment of the outcome of nonsynonymous SNVs with a consensus deleteriousness score, condel.Am. J. Hum. Genet. 2011; 88: 440-449Abstract Full Text Full Text PDF PubMed Scopus (528) Google Scholar, 21Reva B. Antipin Y. Sander C. Predicting the functional impact of protein mutations: application to cancer genomics.Nucleic Acids Res. 2011; 39e118Crossref PubMed Scopus (1077) Google Scholar, 22Choi Y. Sims G.E. Murphy S. Miller J.R. Chan A.P. Predicting the functional effect of amino acid substitutions and indels.PLoS One. 2012; 7e46688Crossref PubMed Scopus (1601) Google Scholar, 23Sim N.-L. Kumar P. Hu J. Henikoff S. Schneider G. Ng P.C. SIFT web server: predicting effects of amino acid substitutions on proteins.Nucleic Acids Res. 2012; 40: W452-W457Crossref PubMed Scopus (979) Google Scholar, 24Shihab H.A. Gough J. Cooper D.N. Stenson P.D. Barker G.L.A. Edwards K.J. Day I.N.M. Gaunt T.R. Predicting the functional, molecular, and phenotypic consequences of amino acid substitutions using hidden Markov models.Hum. Mutat. 2013; 34: 57-65Crossref PubMed Scopus (584) Google Scholar, 25Schwarz J.M. Cooper D.N. Schuelke M. Seelow D. MutationTaster2: mutation prediction for the deep-sequencing age.Nat. Methods. 2014; 11: 361-362Crossref PubMed Scopus (1959) Google Scholar, 26Wong K.-C. Zhang Z. SNPdryad: predicting deleterious non-synonymous human SNPs using only orthologous protein sequences.Bioinformatics. 2014; 30: 1112-1119Crossref PubMed Scopus (50) Google Scholar). Some modern prediction protocols use neural network and machine learning methods to integrate various kinds of information (27Raimondi D. Gazzo A.M. Rooman M. Lenaerts T. Vranken W.F. Multilevel biological characterization of exomic variants at the protein level significantly improves the identification of their deleterious effects.Bioinformatics. 2016; 32: 1797-1804Crossref PubMed Scopus (16) Google Scholar, 28Pejaver V. Mooney S.D. Radivojac P. Missense variant pathogenicity predictors generalize well across a range of function-specific prediction challenges.Hum. Mutat. 2017; 38: 1092-1108Crossref PubMed Scopus (25) Google Scholar, 29Dehghanpoor R. Ricks E. Hursh K. Gunderson S. Farhoodi R. Haspel N. Hutchinson B. Jagodzinski F. Predicting the effect of single and multiple mutations on protein structural stability.Molecules. 2018; 23: 251Crossref Scopus (14) Google Scholar). Molecular dynamics (MD), due to its much higher computational power demands and the lack of experimentally determined 3D structures for many proteins, had seldom been used for the prediction of protein variant functionality. However, with the number of known structures growing quickly, and processor capabilities and computational algorithms improving rapidly, this approach is gaining in popularity (30Kucukkal T.G. Petukh M. Li L. Alexov E. Structural and physico-chemical effects of disease and non-disease nsSNPs on proteins.Curr. Opin. Struct. Biol. 2015; 32: 18-24Crossref PubMed Scopus (96) Google Scholar, 31Sneha P. Doss C.G.P. Molecular dynamics: new frontier in personalized medicine.Adv. Protein Chem. Struct. Biol. 2016; 102: 181-224Crossref PubMed Scopus (70) Google Scholar). For example, MD was used to predict the inhibitor binding with 12 commonly found somatic variants of BCR-ABL1 oncogenic protein kinase (32Gibbons D.L. Pricl S. Posocco P. Laurini E. Fermeglia M. Sun H. Talpaz M. Donato N. Quintás-Cardama A. Molecular dynamics reveal BCR-ABL1 polymutants as a unique mechanism of resistance to PAN-BCR-ABL1 kinase inhibitor therapy.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 3550-3555Crossref PubMed Scopus (60) Google Scholar). In another recent study, structures of 79 naturally occurring nonsynonymous SNPs of human triose phosphate isomerase were analyzed, and some were found to destabilize the protein fold (33Oliver C. Timson D.J. In silico prediction of the effects of mutations in the human triose phosphate isomerase gene: towards a predictive framework for TPI deficiency.Eur. J. Med. Genet. 2017; 60: 289-298Crossref PubMed Scopus (4) Google Scholar). There is little doubt that MD will soon become a powerful approach for the prediction of mutation effects on the protein functionality either on its own or as an important part of combined procedures integrating structural and evolutionary information. In this study, we have developed a semiautomated pipeline to model the structures of somatic mutant variants of proteins found in clinical samples of human tumors and predict the effects of the mutations. As a proof of principle, we have applied the procedure to mutants of 8-oxoguanine DNA glycosylase (OGG1). DNA glycosylases, as many other BER enzymes, modulate the resistance of cancer cells to chemotherapy and radiotherapy. OGG1 initiates the repair of oxidized purines, mostly 8-oxoguanine (oxoG) and formamidopyrimidines. In cell cultures, OGG1 significantly attenuates the toxicity of bleomycin (34Wu M. Zhang Z. Che W. Suppression of a DNA base excision repair gene, hOGG1, increases bleomycin sensitivity of human lung cancer cell line.Toxicol. Appl. Pharmacol. 2008; 228: 395-402Crossref PubMed Scopus (24) Google Scholar), thiotepa (35Kobune M. Xu Y. Baum C. Kelley M.R. Williams D.A. Retrovirus-mediated expression of the base excision repair proteins, formamidopyrimidine DNA glycosylase or human oxoguanine DNA glycosylase, protects hematopoietic cells from N,N′,N″-triethylenethiophosphoramide (thioTEPA)-induced toxicity in vitro and in vivo.Cancer Res. 2001; 61: 5116-5125PubMed Google Scholar, 36Xu Y. Hansen W.K. Rosenquist T.A. Williams D.A. Limp-Foster M. Kelley M.R. Protection of mammalian cells against chemotherapeutic agents thiotepa, 1,3-N,N′-bis(2-chloroethyl)-N-nitrosourea, and mafosfamide using the DNA base excision repair genes Fpg and α-hOgg1: implications for protective gene therapy applications.J. Pharmacol. Exp. Ther. 2001; 296: 825-831PubMed Google Scholar), carmustine (36Xu Y. Hansen W.K. Rosenquist T.A. Williams D.A. Limp-Foster M. Kelley M.R. Protection of mammalian cells against chemotherapeutic agents thiotepa, 1,3-N,N′-bis(2-chloroethyl)-N-nitrosourea, and mafosfamide using the DNA base excision repair genes Fpg and α-hOgg1: implications for protective gene therapy applications.J. Pharmacol. Exp. Ther. 2001; 296: 825-831PubMed Google Scholar, 37He Y.-H. Xu Y. Kobune M. Wu M. Kelley M.R. Martin II, W.J. Escherichia coli FPG and human OGG1 reduce DNA damage and cytotoxicity by BCNU in human lung cells.Am. J. Physiol. Lung Cell Mol. Physiol. 2002; 282: L50-L55Crossref PubMed Scopus (42) Google Scholar), mafosfamide (36Xu Y. Hansen W.K. Rosenquist T.A. Williams D.A. Limp-Foster M. Kelley M.R. Protection of mammalian cells against chemotherapeutic agents thiotepa, 1,3-N,N′-bis(2-chloroethyl)-N-nitrosourea, and mafosfamide using the DNA base excision repair genes Fpg and α-hOgg1: implications for protective gene therapy applications.J. Pharmacol. Exp. Ther. 2001; 296: 825-831PubMed Google Scholar), cisplatin, and oxaliplatin (38Preston T.J. Henderson J.T. McCallum G.P. Wells P.G. Base excision repair of reactive oxygen species-initiated 7,8-dihydro-8-oxo-2′-deoxyguanosine inhibits the cytotoxicity of platinum anticancer drugs.Mol. Cancer Ther. 2009; 8: 2015-2026Crossref PubMed Scopus (45) Google Scholar). Thus, somatic mutations in the OGG1 gene in tumor cells could affect their sensitivity to therapeutic interventions. Using the computational pipeline, we have identified three novel clinical variants of OGG1 with impaired activity. At the beginning of this study, the Catalogue Of Somatic Mutations In Cancer (COSMIC) database (39Tate J.G. Bamford S. Jubb H.C. Sondka Z. Beare D.M. Bindal N. Boutselakis H. Cole C.G. Creatore C. Dawson E. Fish P. Harsha B. Hathaway C. Jupe S.C. Kok C.Y. et al.COSMIC: the catalogue of somatic mutations in cancer.Nucleic Acids Res. 2019; 47: D941-D947Crossref PubMed Scopus (944) Google Scholar) held 39 nonsynonymous single-nucleotide mutations in the OGG1 gene. We have used four popular software packages for mutation effect prediction from sequence conservation—SIFT (18Kumar P. Henikoff S. Ng P.C. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm.Nat. Protoc. 2009; 4: 1073-1081Crossref PubMed Scopus (4557) Google Scholar), FATHMM (24Shihab H.A. Gough J. Cooper D.N. Stenson P.D. Barker G.L.A. Edwards K.J. Day I.N.M. Gaunt T.R. Predicting the functional, molecular, and phenotypic consequences of amino acid substitutions using hidden Markov models.Hum. Mutat. 2013; 34: 57-65Crossref PubMed Scopus (584) Google Scholar), MutationTaster (25Schwarz J.M. Cooper D.N. Schuelke M. Seelow D. MutationTaster2: mutation prediction for the deep-sequencing age.Nat. Methods. 2014; 11: 361-362Crossref PubMed Scopus (1959) Google Scholar), and PROVEAN (22Choi Y. Sims G.E. Murphy S. Miller J.R. Chan A.P. Predicting the functional effect of amino acid substitutions and indels.PLoS One. 2012; 7e46688Crossref PubMed Scopus (1601) Google Scholar)—to analyze the functionality of these mutations. All of them produce a binary output: whether the protein function is expected to be affected or not affected in some way. Of 39 substitutions, 36 were included and three (all located in exon 8, the last exon of the human gene) were discarded because of the insufficient number of homologous sequences (Table S1). Cohen's κ coefficient (40McHugh M.L. Interrater reliability: the kappa statistic.Biochem. Med. 2012; 22: 276-282Crossref PubMed Google Scholar) was used to measure the agreement between the prediction methods (−1 ≤ κ ≤ 1; where κ = −1 for complete disagreement, κ = 1 for complete agreement, and κ = 0 for agreement expected from random coincidence). As can be seen from Table 1, the agreement between different algorithms was rather low in most cases. For a comparison, we took the gene for another human DNA glycosylase, UNG, and repeated the analysis for nonsynonymous single-nucleotide mutations found in the COSMIC database its coding region (44 total, all included; Table S1). Neither the UNG mutation set nor the combined OGG1/UNG set demonstrated better performance than the OGG1 set (Table 1). Given the discordance in the predictions by sequence-based algorithms, we set out to apply MD for estimating the mutation effects in the OGG1 coding sequence.Table 1Cohen's κ for agreement between the predictions of the effect of mutations in OGG1 and UNG by different algorithmsFATHMMMutationTasterPROVEANSIFT0.163aFirst number is Cohen's κ for the OGG1 mutation set, second, for the UNG mutation set, third, for the combined set.0.3850.7800.0680.3230.1780.0870.4280.490FATHMM0.1920.2890.0490.1170.0790.177MutationTaster0.5030.3710.464a First number is Cohen's κ for the OGG1 mutation set, second, for the UNG mutation set, third, for the combined set. Open table in a new tab We have designed a computational pipeline that retrieves all missense variants of the gene of interest from the COSMIC database (or, with an appropriate setup of the connection module, from any database that can be automatically parsed), maps them on the selected protein sequence and structure, randomly selects a given number of mutants, and prepares them for MD. The script is available as an open source code from https://doi.org/10.5281/zenodo.3828057. Mapping is an important part of the procedure because many protein isoforms are known for OGG1 (41Kohno T. Shinmura K. Tosaka M. Tani M. Kim S.-R. Sugimura H. Nohmi T. Kasai H. Yokota J. Genetic polymorphisms and alternative splicing of the hOGG1 gene, that is involved in the repair of 8-hydroxyguanine in damaged DNA.Oncogene. 1998; 16: 3219-3225Crossref PubMed Scopus (412) Google Scholar, 42Boldinova E.O. Khairullin R.F. Makarova A.V. Zharkov D.O. Isoforms of base excision repair enzymes produced by alternative splicing.Int. J. Mol. Sci. 2019; 20: 3279Crossref Scopus (3) Google Scholar), and the COSMIC database uses the longest polypeptide, the major mitochondrial isoform OGG1-2a, as a reference, whereas the vast majority of biochemical and all structural data in the literature are for the shorter major nuclear isoform OGG1-1a. Of the randomly selected 20 mutants, only one (R229Q) turned out to be studied before with respect to its biochemical properties, and for another one (R131G), a substitution of Gln for the same Arg131 residue was characterized as inactive (Table 2). The location of the mutants in the sequence and structure of OGG1 protein is shown in Figure 1. All 20 mutants and WT OGG1 were subjected to MD in explicit water.Table 2Somatic mutants of OGG1 investigated in this studyOGG1 variantPredicted effectaBinary output from individual algorithms, coded as the following: Yes: SIFT "affects protein function," FATHMM "damaging," MutationTaster "disease causing," PROVEAN "deleterious"; No: SIFT "tolerated," FATHMM "tolerated," MutationTaster "polymorphism," PROVEAN "neutral." Combined score: sum of the four prediction, with +1 for "No" and −1 for "Yes"; negative values indicate an effect on the protein function.Properties reported in the literatureSIFTFATHMMMutationTasterPROVEANCombined scoreExperimentalR87KNoNoNoNo+4P88LNoNoYesNo+2E92DNoNoYesNo+2NoR97CYesNoNoYes0H111RNoNoNoNo+4NoS115FYesNoNoYes0S118FYesNoYesYes−2NoR131GYesYesYesYes−4R131Q: small-cell lung carcinoma; inactive (86Chevillard S. Radicella J.P. Levalois C. Lebeau J. Poupon M.-F. Oudard S. Dutrillaux B. Boiteux S. Mutations in OGG1, a gene involved in the repair of oxidative DNA damage, are found in human lung and kidney tumours.Oncogene. 1998; 16: 3083-3086Crossref PubMed Scopus (208) Google Scholar, 87Anderson P.C. Daggett V. The R46Q, R131Q and R154H polymorphs of human DNA glycosylase/β-lyase hOgg1 severely distort the active site and DNA recognition site but do not cause unfolding.J. Am. Chem. Soc. 2009; 131: 9506-9515Crossref PubMed Scopus (18) Google Scholar)I145MYesNoYesYes−2YesA153TNoYesNoNo+2R161WYesYesYesYes−4Yes/NoG202CYesYesYesYes−4YesR206CYesYesYesYes−4NoQ226HNoNoYesNo+2NoR229QNoNoNoYes+2Leukemia; active but thermolabile at 37 °C; cells radiation sensitive (88Hyun J.-W. Choi J.-Y. Zeng H.-H. Lee Y.-S. Kim H.-S. Yoon S.-H. Chung M.-H. Leukemic cell line, KG-1 has a functional loss of hOGG1 enzyme due to a point mutation and 8-hydroxydeoxyguanosine can kill KG-1.Oncogene. 2000; 19: 4476-4479Crossref PubMed Scopus (49) Google Scholar, 89Hyun J.-W. Cheon G.-J. Kim H.-S. Lee Y.-S. Choi E.-Y. Yoon B.-H. Kim J.-S. Chung M.-H. Radiation sensitivity depends on OGG1 activity status in human leukemia cell lines.Free Radic. Biol. Med. 2002; 32: 212-220Crossref PubMed Scopus (38) Google Scholar, 90Hill J.W. Evans M.K. A novel R229Q OGG1 polymorphism results in a thermolabile enzyme that sensitizes KG-1 leukemia cells to DNA damaging agents.Cancer Detect. Prev. 2007; 31: 237-243Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar)Q263HYesNoYesNo0NoV267MYesNoYesYes−2YesT285MNoNoNoNo+4NoP291QYesNoNoNo+2NoS292NNoNoNoNo+4Yes/Noa Binary output from individual algorithms, coded as the following: Yes: SIFT "affects protein function," FATHMM "damaging," MutationTaster "disease causing," PROVEAN "deleterious"; No: SIFT "tolerated," FATHMM "tolerated," MutationTaster "polymorphism," PROVEAN "neutral." Combined score: sum of the four prediction, with +1 for "No" and −1 for "Yes"; negative values indicate an effect on the protein function. Open table in a new tab Forty ns trajectories were essentially equilibrated after 20 ns (Fig. S1), so the last 20 ns were analyzed. To extract functionally relevant differences between the mutants, we focused on the enzyme's active site, defined as all residues in protein and DNA, at least one atom of which is within 5 Å of 8-oxo-2′-deoxyguanosine (oxodG) in the OGG1–DNA complex X-ray structure (1EBM (43Bruner S.D. Norman D.P.G. Verdine G.L. Structural basis for recognition and repair of the endogenous mutagen 8-oxoguanine in DNA.Nature. 2000; 403: 859-866Crossref PubMed Scopus (773) Google Scholar)) that served as a starting point for our models. Thus defined, the active site encompasses 23 amino acid residues and three nucleotides: oxodG and its 5′ and 3′ neighbors. Of all studied mutations, only V267M affected a residue in the active site, which includes N and Cα atoms of Val267. In addition, we analyzed a narrower set of key geometric parameters for the catalytic dyad Lys249–Asp268, namely the Nζ[K249]–C1′[oxodG] and Oδ1/Oδ2[D268]–O4′[oxodG] distances and the Nζ[K249]–C1′[oxodG]–N9[oxodG] angle. These three parameters reflect correct positioning of the catalytic amine nucleophile of Lys249 and the damaged nucleotide (43Bruner S.D. Norman D.P.G. Verdine G.L. Structural basis for recognition and repair of the endogenous mutagen 8-oxoguanine in DNA.Nature. 2000; 403: 859-866Crossref PubMed Scopus (773) Google Scholar, 44Norman D.P.G. Chung S.J. Verdine G.L. Structural and biochemical exploration of a critical amino acid in human 8-oxoguanine glycosylase.Biochemistry. 2003; 42: 1564-1572Crossref PubMed Scopus (100) Google Scholar) and were earlier shown to be good predictors for the OGG1 activity on a limited set of substrates (oxoG:C, oxoG:A, and AP:C) and active site–obstructing mutants (45Lukina M.V. Popov A.V. Koval V.V. Vorobjev Y.N. Fedorova O.S. Zharkov D.O. DNA damage processing by human 8-oxoguanine-DNA glycosylase mutants with the occluded active site.J. Biol. Chem. 2013; 288: 28936-28947Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 46Popov A.V. Yudkina A.V. Vorobjev Y.N. Zharkov D.O. Catalytically competent conformation of the active site of human 8-oxoguanine–DNA glycosylase.Biochemistry (Mosc). 2020; 85: 192-204Crossref PubMed Scopus (2) Google Scholar). Principal component analysis (PCA) is often used to reduce dimensionality of multivariate data with minimal information loss. We have first performed the PCA using the dihedral covariance matrix taking into account all torsion angles of the active-site residues for all analyzed variants. Principal components 1 and 2 for all OGG1 mutants are illustrated in Figure 2A. To cluster the full trajectories rather than their means (e.g., as presented in Fig. 2D), we have calculated Bhattacharya similarity coefficients in the PC1 versus PC2 plane for all pairs of trajectories (see Experimental procedures) and presented the results as a tree (Fig. 2G). The variants formed several tight clusters; P88L and R229Q were closest to the WT enzyme, and I145M was close to this group. Q263H and P291Q lied farthest from WT OGG1 and were wide