Structural insights into the interactions and epigenetic functions of human nucleic acid repair protein ALKBH6

Abstract

Human AlkB homolog 6, ALKBH6, plays key roles in nucleic acid damage repair and tumor therapy. However, no precise structural and functional information are available for this protein. In this study, we determined atomic resolution crystal structures of human holo-ALKBH6 and its complex with ligands. AlkB members bind nucleic acids by NRLs (nucleotide recognition lids, also called Flips), which can recognize DNA/RNA and flip methylated lesions. We found that ALKBH6 has unusual Flip1 and Flip2 domains, distinct from other AlkB family members both in sequence and conformation. Moreover, we show that its unique Flip3 domain has multiple unreported functions, such as discriminating against double-stranded nucleic acids, blocking the active center, binding other proteins, and in suppressing tumor growth. Structural analyses and substrate screening reveal how ALKBH6 discriminates between different types of nucleic acids and may also function as a nucleic acid demethylase. Structure-based interacting partner screening not only uncovered an unidentified interaction of transcription repressor ZMYND11 and ALKBH6 in tumor suppression but also revealed cross talk between histone modification and nucleic acid modification in epigenetic regulation. Taken together, these results shed light on the molecular mechanism underlying ALKBH6-associated nucleic acid damage repair and tumor therapy. Human AlkB homolog 6, ALKBH6, plays key roles in nucleic acid damage repair and tumor therapy. However, no precise structural and functional information are available for this protein. In this study, we determined atomic resolution crystal structures of human holo-ALKBH6 and its complex with ligands. AlkB members bind nucleic acids by NRLs (nucleotide recognition lids, also called Flips), which can recognize DNA/RNA and flip methylated lesions. We found that ALKBH6 has unusual Flip1 and Flip2 domains, distinct from other AlkB family members both in sequence and conformation. Moreover, we show that its unique Flip3 domain has multiple unreported functions, such as discriminating against double-stranded nucleic acids, blocking the active center, binding other proteins, and in suppressing tumor growth. Structural analyses and substrate screening reveal how ALKBH6 discriminates between different types of nucleic acids and may also function as a nucleic acid demethylase. Structure-based interacting partner screening not only uncovered an unidentified interaction of transcription repressor ZMYND11 and ALKBH6 in tumor suppression but also revealed cross talk between histone modification and nucleic acid modification in epigenetic regulation. Taken together, these results shed light on the molecular mechanism underlying ALKBH6-associated nucleic acid damage repair and tumor therapy. As α-ketoglutarate (α-KG) and Fe(II)-dependent dioxygenases, AlkB family members are widespread in the biological kingdom and play important roles in epigenetics. The AlkB gene was found in Escherichia coli, and its product was involved in repairing alkylated bases (1Kataoka H. Yamamoto Y. Sekiguchi M. A new gene (alkB) of Escherichia coli that controls sensitivity to methyl methane sulfonate.J. Bacteriol. 1983; 153: 1301-1307Google Scholar). Human genome encodes nine AlkB homologs named ALKBH1–8 and FTO (fat mass and obesity-associated) with low sequence identity (2Kurowski M.A. Bhagwat A.S. Papaj G. Bujnicki J.M. Phylogenomic identification of five new human homologs of the DNA repair enzyme AlkB.BMC Genomics. 2003; 4: 48Google Scholar, 3Gerken T. Girard C.A. Tung Y.C. Webby C.J. Saudek V. Hewitson K.S. Yeo G.S. McDonough M.A. Cunliffe S. McNeill L.A. Galvanovskis J. Rorsman P. Robins P. Prieur X. Coll A.P. et al.The obesity-associated FTO gene encodes a 2-oxoglutarate-dependent nucleic acid demethylase.Science. 2007; 318: 1469-1472Google Scholar). All the nine AlkB homologs have different substrates and functions including DNA repair, RNA modification, actomyosin demethylation, and fatty acid metabolism (4Falnes P.O. Repair of 3-methylthymine and 1-methylguanine lesions by bacterial and human AlkB proteins.Nucleic Acids Res. 2004; 32: 6260-6267Google Scholar, 5Lee D.H. Jin S.G. Cai S. Chen Y. Pfeifer G.P. O'Connor T.R. Repair of methylation damage in DNA and RNA by mammalian AlkB homologues.J. Biol. Chem. 2005; 280: 39448-39459Google Scholar, 6Li M.-M. Nilsen A. Shi Y. Fusser M. Ding Y.-H. Fu Y. Liu B. Niu Y. Wu Y.-S. Huang C.-M. Olofsson M. Jin K.-X. Lv Y. Xu X.-Z. He C. et al.ALKBH4-dependent demethylation of actin regulates actomyosin dynamics.Nat. Commun. 2013; 4: 1832Google Scholar). Most of them have essential roles in metabolism and diseases curbing such as cancers (7Xie Q. Wu T.P. Gimple R.C. Li Z. Prager B.C. Wu Q. Yu Y. Wang P. Wang Y. Gorkin D.U. Zhang C. Dowiak A.V. Lin K. Zeng C. Sui Y. et al.N(6)-methyladenine DNA modification in glioblastoma.Cell. 2018; 175: 1228-1243.e20Google Scholar, 8Xiao C.L. Zhu S. He M. Chen D. Zhang Q. Chen Y. Yu G. Liu J. Xie S.Q. Luo F. Liang Z. Wang D.P. Bo X.C. Gu X.F. Wang K. et al.N(6)-Methyladenine DNA modification in the human genome.Mol. 2018; Scholar, Li T. Wang Q. Li Y. Yu K. Wang M. Lin Luo Wang Lin Liu Q. Wu et by Scholar). of nucleic acids methylated have as AlkB members substrates G.S. M. of AlkB nucleic acid new epigenetic Chem. Scholar). The AlkB family members have substrates can DNA modification in human (7Xie Q. Wu T.P. Gimple R.C. Li Z. Prager B.C. Wu Q. Yu Y. Wang P. Wang Y. Gorkin D.U. Zhang C. Dowiak A.V. Lin K. Zeng C. Sui Y. et al.N(6)-methyladenine DNA modification in glioblastoma.Cell. 2018; 175: 1228-1243.e20Google also can et B. M. G. M. Human AlkB homolog a that in DNA and Biol. Chem. Scholar, F. Luo G. Wang X. Fu Y. J. Wang X. Z. Q. G. H. D. M. A. T. et demethylation regulates Scholar). and both in and and in T. P. T. B. of DNA damage by human S. A. Scholar). structural can on and also on actin M.-M. Nilsen A. Shi Y. Fusser M. Ding Y.-H. Fu Y. Liu B. Niu Y. Wu Y.-S. Huang C.-M. Olofsson M. Jin K.-X. Lv Y. Xu X.-Z. He C. et al.ALKBH4-dependent demethylation of actin regulates actomyosin dynamics.Nat. Commun. 2013; 4: 1832Google Scholar). and can RNA G.S. M. of AlkB nucleic acid new epigenetic Chem. Scholar). to to and of S. Liu C. J. C. Zhang Q. X. Z. Wang Y. X. Wang et demethylation regulates RNA Biol. Scholar). FTO to RNA can with a in an which its functions Li T. Wang Q. Li Y. Yu K. Wang M. Lin Luo Wang Lin Liu Q. Wu et by Scholar). we structures of human and C. Liu Y. Wang G. Z. Zhang Q. Wu Y. C. Chen Z. structures of the human RNA reveal for substrate Biol. Chem. Scholar, G. He Q. C. Liu Y. Z. X. Wu P. Chen Z. The atomic resolution of human AlkB homolog a key for and Biol. Chem. Scholar, Liu Chen Q. Wu Chen Z. Structural of nucleic acid recognition and demethylation by human Res. precise structural information for function a of the AlkB ALKBH6 in and with the in and (2Kurowski M.A. Bhagwat A.S. Papaj G. Bujnicki J.M. Phylogenomic identification of five new human homologs of the DNA repair enzyme AlkB.BMC Genomics. 2003; 4: 48Google Scholar, K. K. K. A. H. T. M. Y. T. Yamamoto H. and of human family 2007; Scholar). of ALKBH6 in but not in that ALKBH6 may play important roles in C. Li D. J. J. Zhang Chen Y. X. Y. H. Zhang Li F. and in with by J. Scholar). with a of ALKBH6 can tumor the F. J. S. A.P. T. D. J. A. and of new to Scholar). ALKBH6 has as a in P. L.A. T.R. M. G. and of Scholar). that ALKBH6 can coli against methyl methane S. A. D. Human ALKBH6 for of and of in Scholar). to the ALKBH6 plays a in from DNA damage and the of ALKBH6 in with has to S. A. D. Human ALKBH6 for of and of in Scholar). The information that ALKBH6 may play key roles in DNA damage repair and tumor therapy. et H. of a RNA ALKBH6 in and J. that ALKBH6, with identity to human ALKBH6, and as a of RNA the of ALKBH6 was that ALKBH6 in J. S. H. RNA methylation in and Scholar). However, the interacting and of human ALKBH6 to Moreover, ALKBH6 has a low sequence identity to the of its In this we structures of human ALKBH6, in complex with structures reveal a with multiple unique multiple of nucleic acids we that ALKBH6 bind nucleic acids and nucleic Structure-based interacting partner screening found that ALKBH6 with both and which for functional The human ALKBH6 from coli to crystal screening both and the we the ALKBH6 and crystal of ALKBH6 in complex with and However, to the low sequence to structures in the molecular multiple structures as we different of the by M. Wang H. Wang S. J. Wang Z. H. Xu J. the as molecular such as of different resolution and the was found by F. P. A Biol. Scholar). of and the of the was to a resolution of in the with an of and an of The of ALKBH6 with and was determined by molecular the as a and was to a of and of ALKBH6 of holo-ALKBH6 and structures revealed in the with a of and of ALKBH6 for resolution of of of in and of the in and of the for resolution in and of the in a new The of ALKBH6 a double-stranded the which in the M.A. C. C.J. Structural on human Biol. Scholar). The in the that S. the structural homolog of the ALKBH6 in the with the of and an of The ALKBH6 also has with other human AlkB family such as of and of of and of and of and of in the structures of the such as the and the the active In ALKBH6, the which by The a by and a by and and and the active and and and the with other AlkB family ALKBH6 also has Flip1 from to and Flip2 from to a unique from to including a between and in the to named Flip3 However, in the of the Flip3 from to no not The may that the from the the of the Flip3 in the crystal from to between and no in structures and not the these and from the active with and an conformation. and the between them with the as in the In a M.A. C. C.J. Structural on human Biol. the in the active of the in ALKBH6, and the of and the In the active of the an of from the was found which not in other AlkB family members which and with and of In the the in a that in other and to the of and from by are between and ALKBH6 with in the the key in the interaction have no between the and the of can the binding of to ALKBH6 was to have substrates M.-M. Nilsen A. Shi Y. Fusser M. Ding Y.-H. Fu Y. Liu B. Niu Y. Wu Y.-S. Huang C.-M. Olofsson M. Jin K.-X. Lv Y. Xu X.-Z. He C. et al.ALKBH4-dependent demethylation of actin regulates actomyosin dynamics.Nat. Commun. 2013; 4: 1832Google human AlkB family members nucleic acid was to to and of S. Liu C. J. C. Zhang Q. X. Z. Wang Y. X. Wang et demethylation regulates RNA Biol. Scholar). However, the of ALKBH6 a for Structural analyses of structures of the domain the between ALKBH6 and other AlkB proteins, we AlkB family bind and substrates by recognition also called Flips), key the domain B. Liu D. Wang Z. Y. on key and Scholar). The NRLs to B. Liu D. Wang Z. Y. on key and Scholar, G. AlkB flip in different and are AlkB The Flip1 and Flip2 of ALKBH6 the domain and are different from of other human AlkB family and The Flip1 of ALKBH6 a a and a the the family which may The the of the and in the to the of Flip1 may a substrate In the complex which in the Flip1 of the and the DNA C. X. He C. structures of DNA/RNA repair AlkB and to ALKBH6 in the The Flip2 of ALKBH6 as a the human AlkB family proteins, only the Flip2 of ALKBH6 not have In the of Flip1 and only Flip1 and Flip2 of ALKBH6 have a acids with Moreover, a of the domain that Flip2 by the Flip2 has interaction with the ALKBH6 has the of the family an by from Flip2 and from Moreover, the of with of and from The of with the of the of Flip2 and the of Flip2 that of Flip1 As the substrate for ALKBH6 we the and with The of the of ALKBH6 different from that of the other family ALKBH6 has with the of the active has to and A the active between Flip1 and The may bind the Most of the AlkB family members have to substrates the active However, to the substrates of ALKBH6 of these As the in the binding we of these involved in In the of the complex C. X. He C. structures of DNA/RNA repair AlkB and to the of interaction with the in the in In structures of the human AlkB family the of Flip1 in has a key that involved in interacting with the nucleic acid A and in the C. X. He C. structures of DNA/RNA repair AlkB and to ALKBH6 and are in C. F. M. and RNA binding of the RNA recognition and AlkB in human AlkB homolog an enzyme Biol. Chem. Scholar). In the of and are ALKBH6, and Moreover, the ALKBH6 active and that are to in these reveal that ALKBH6 as a DNA/RNA other human AlkB family As of ALKBH6 in coli methyl methane S. A. D. Human ALKBH6 for of and of in Scholar). on the structural we that ALKBH6 with nucleic acids we such as and DNA with different of in the of Liu Chen Q. Wu Chen Z. Structural of nucleic acid recognition and demethylation by human Res. Scholar, M. S. Liu G. Li Z. Liu X. Wu T. A. Li H. as an of Res. Scholar). and multiple we found that ALKBH6 bind and and DNA and DNA and RNA and but not All the nucleic acids have no such as the an was to the the complex In ALKBH6 to and RNA H. of a RNA ALKBH6 in and J. but the has not Human ALKBH6 has a identity with its in that human ALKBH6 bind We as a substrate to with We a binding as by and a complex in In the has a the results to ALKBH6 bind to and this the to the binding with a binding and the has a on binding the key in binding nucleic acids, we to the essential for the binding the was with the and As both and are in Flip2 plays key roles substrates to the and binding between ALKBH6 and nucleic acid of the nucleic acid the by Flip2 and and the other the by Flip2 and Flip1 bind nucleic the other with which has and no a the of these a for the molecular mechanism of ALKBH6 binding to substrates and has an to the recognition to the in the human AlkB ALKBH6 has the with which can RNA (2Kurowski M.A. Bhagwat A.S. Papaj G. Bujnicki J.M. Phylogenomic identification of five new human homologs of the DNA repair enzyme AlkB.BMC Genomics. 2003; 4: 48Google Scholar, C. F. M. and RNA binding of the RNA recognition and AlkB in human AlkB homolog an enzyme Biol. Chem. Scholar). A also that the structural homolog of ALKBH6 human AlkB family We ALKBH6 and the unique Flip3 of ALKBH6 has a with the RNA recognition of Flip3 has a function as we an Flip3 was the binding of this to was that of the Flip3 may have a on the binding of ALKBH6 to nucleic acids, to the function of the we the structures of this In the human AlkB only ALKBH6, and have a Flip3 and The Flip3 to the of the methylated with and to C. X. He C. structures of DNA/RNA repair AlkB and to Scholar). The Flip3 to the of the by a between and which the binding of the and only substrates C. Liu Y. Wang G. Z. Zhang Q. Wu Y. C. Chen Z. structures of the human RNA reveal for substrate Biol. Chem. Scholar). ALKBH6 has the Flip3 this are in of As we the the The the Flip3 to the complex structures are with ALKBH6, the methylated by ALKBH6, the has a with the Flip3 Moreover, in Flip3 a and the of the The may its that the of Flip3 can the binding to the we the binding of the to results that this bind and the binding was and Taken together, Flip3 of ALKBH6 the of double-stranded nucleic acids to the active and may ALKBH6 the to against double-stranded nucleic of with a of ALKBH6 which the complex and the can tumor F. J. S. A.P. T. D. J. A. and of new to Scholar). As the unique Flip3 has the in the and from the we to bind on the of the we found that Flip3 the ALKBH6 active and we that ALKBH6 with Flip3 from we by and We the and in the human that with ALKBH6, we the human on the sequence to are we the and only found ALKBH6 no for and we as only found on the structural information that are in the of the active of ALKBH6 the binding by we the by to and A of and We to to and In the with not bind a from to of ZMYND11 found with with ALKBH6 by and the binding was Moreover, the binding of ZMYND11 to ALKBH6 was ALKBH6 a new the ALKBH6 function by As the of ZMYND11 has a that to the we ALKBH6 can bind other a an also has a that to the from to sequence not show to the Flip3 of we a from to of its domain and the interaction between ALKBH6 and by As the results in ALKBH6 no interaction with the the interaction between ALKBH6 and the ZMYND11 domain has sequence has of in human diseases and Z. A. A. Y. J. C.J. V. et for and of J. 2018; Scholar, Y. K. T. M. T. and for and in Scholar). AlkB family members can in the modification of nucleic acids and have on human diseases C.L. Zhu S. He M. Chen D. Zhang Q. Chen Y. Yu G. Liu J. Xie S.Q. Luo F. Liang Z. Wang D.P. Bo X.C. Gu X.F. Wang K. et al.N(6)-Methyladenine DNA modification in the human genome.Mol. 2018; Scholar, J. Wang J. Gu Q. Y. Y. Zhu J. Zhang Q. The biological function of and its in human Scholar, Liu Chen Liu of homologs for Scholar, Z. M. B. Luo G. Wu Y. Li J. Z. Z. Q. Wang H. RNA of Acids Res. Scholar, T. M. D. M. K. A. A. D. D. A. J. J. in and for Scholar). a of the AlkB ALKBH6 has a for we the and interacting of ALKBH6 and for functional In this study, we found that ALKBH6 bind in the active which has found in other members of this in the of and may with the and in the functional of that the in the active in a the of this the of a that the in the AlkB may a for the against AlkB family As human AlkB homologs to on with a on an RNA C. Liu Y. Wang G. Z. Zhang Q. Wu Y. C. Chen Z. structures of the human RNA reveal for substrate Biol. Chem. Scholar, Y. Chen He C. and of the DNA repair AlkB from Escherichia Chem. 2004; Scholar, Y. He C. of human and bacterial AlkB with DNA as Acids Res. 2004; 32: Scholar, M. M. of the RNA that the in human Scholar). In the human AlkB DNA Liu Chen Q. Wu Chen Z. Structural of nucleic acid recognition and demethylation by human Res. Scholar, M. V. F. G. Human and key for demethylation to DNA/RNA J. as substrate C. X. He C. structures of DNA/RNA repair AlkB and to RNA as substrates Z. M. B. Luo G. Wu Y. Li J. Z. Z. Q. Wang H. RNA of Acids Res. Scholar, M. V. F. G. Human and key for demethylation to DNA/RNA J. and and FTO function as RNA G. Niu Y. P. Huang C.-M. Li C.J. Shi Y. Wang Z. Q. X. et a mammalian RNA that RNA metabolism and 2013; Scholar, D. Human AlkB homolog a for modification and DNA damage Biol. Scholar, G. Fu Y. X. Q. G. Y. C. T. T. He C. in RNA a substrate of the obesity-associated Chem. Biol. Scholar). In study, we found that the unique Flip3 was key for the binding of ALKBH6 to nucleic acids Moreover, ALKBH6 to other types of sequence of in ALKBH6 was M. V. F. G. Human and key for demethylation to DNA/RNA J. that ALKBH6 has We not of ALKBH6 on of nucleic acids, such as and Structural reveal that ALKBH6 has unique the Flip1 of ALKBH6 the the family members and an to in to the and the DNA C. X. He C. structures of DNA/RNA repair AlkB and to in the Flip2 of AlkB and from in and in with substrates by the ALKBH6 Flip2 ALKBH6 has the as its Flip3 has the and the active by the Flip3 Structure-based sequence that the ALKBH6 Flip3 in human a unique with other family members that in other and In a of and Flip2 results in ALKBH6 and binding to this may the to the substrates of ALKBH6 In a study, et S. M. A. S. P.O. methylation of by a of a human Commun. to and the binding partner of the from the crystal of the we found that the unique Flip3 the active with a of The interaction between Flip3 and ALKBH6 may by the in Flip3 and the active and we the Z. Li X. Li M. Wu Zhang M. C. Li Z. Liu Y. Chen Z. M. C. Y. S. et structures of and its complex with DNA reveal recognition with the results that ALKBH6 was in However, the was the was the of to interacting of ALKBH6, we sequence to the human and found revealed that ALKBH6 not only with but also has a binding to new for functional ZMYND11 can histone to transcription and tumor suppression H. Li Y. Y. S. S. D. K. Y. Z. Wu J. Li B. Li Li H. Shi X. ZMYND11 histone to transcription and Scholar). ZMYND11 has for including a a and a domain for a The from to the domain to the domain the binding between ZMYND11 and can H. Li Y. Y. S. S. D. K. Y. Z. Wu J. Li B. Li Li H. Shi X. ZMYND11 histone to transcription and Scholar). In the we found that ALKBH6 bind nucleic results that also with proteins, the of ALKBH6 in We that the binding of ZMYND11 may the of ALKBH6 and the demethylation of nucleic acids, no has as a for with ALKBH6 and may functions in the of the revealed that ALKBH6 in and Flip3 to the and to We on the and the interaction between ALKBH6 and The in Flip2 can the interaction between ALKBH6 and have to the ALKBH6 by the of the of results that the of the key the interaction between ALKBH6 and the of ALKBH6 in tumor we structures of ALKBH6, which the structural of the AlkB on the structural information we that ALKBH6 can bind different types of nucleic we the key for its binding new its substrates and for its We also found that ALKBH6 with ZMYND11 which may as a new of for ALKBH6 function In the results the bases for ALKBH6 functions such as nucleic acid damage repair and tumor therapy. The ALKBH6 was from the was and in a with a by a was as a to show the of which can the screening P. with in the to and with for an ALKBH6, and a with a The the and by DNA and in to was by the of The proteins, by T. J. J. Li Y. K. Huang M. P. of an to by and for and and coli by for and in A and by P. for to the was to a the was with and with was to the to the a The was a to and The was an and with a from to in The was by with and by by The was by to to for All the The for and was in of both and with of ALKBH6 from P. in the we the of ALKBH6, different and both and for ALKBH6 was with a the was with and on for was to The to a for and in The on and the and with of Z. of in Scholar). are in ALKBH6 has a low sequence identity to the structures in the we to a molecular for the a new to and we a of ALKBH6 by M. Wang H. Wang S. J. Wang Z. H. Xu J. the as an We different of the as such as of different resolution and the was found by F. P. A Biol. with an of the both the and in the the P. B. K. and of Biol. the was to the by The has a with and with an of and an of we and the of the and 2007; the P. A. et of the and Biol. and in the G. Chen et A for Biol. Scholar). to the the and the by P. F. for the of crystal Biol. of and the an of The complex of was determined by molecular the as a with for Biol. Scholar). The has a of and a of The has a of with a of was to the for the interaction of with proteins, and DNA the binding between and ALKBH6, ALKBH6 and with a to the of in was with for the was for with and the the binding between DNA and ALKBH6 the with the was by The was We by DNA with with The are for and for with ALKBH6 was to interaction in and with nucleic acid from nucleic acid was to the nucleic acid binding of ALKBH6 with the to was with DNA in for was to and of the was on a to in for The was with the of to the of ALKBH6 was with and the interacting of was with different for in binding and in the of the was with the to the and on the by and by of on the by in a of from to by D. B. a for and Scholar). The was and and for the crystal structures have with the and information S. Y. Y. C. M. M. H. S. S. S. M. T. T. H. A. et and unique of in to Scholar, M. S. Y. A. M. Y. C. M. K. K. et of in Scholar, X. B. F. G. V. T. A. K. et new and in Scholar, S. Li J. H. C. X. X. Li Y. Li Q. Wang H. J. G. Wu M. Ding C. Chen et of Commun. Scholar, Z. C. Li X. Luo B. T. Zhang X. H. Wang J. Z. Chen Y. J. S. Y. M. et al.The of in 2018; Scholar, Yu J. H. et of Res. Scholar, M. Y. P. A. G. M. S. D. K. A. et in Scholar, C. F. B. D. A. C. M. D. G. G. J. P. G. of from in to Scholar, C. C. S. K. A. et in to Scholar, M. X. M. J. K. G. G. et in and Scholar, M.A. K. H. H. V. S. C. et of Scholar, P. K. C. P. K. J.M. B. C. et al.The of as the of J. Scholar, Wang J. Liu Z. P. J. Chen A. et in Scholar, S. J. B. of to for and with Scholar, A.P. S. M. A. K.S. et Scholar, K. F. C. V. F. L.A. D. A. G. C. M. et the molecular of in an of and Scholar, S. C. Wang J. C. K. et of in Scholar, S. T. M. K. Wu X. Girard Y. Chen et for of in 2018; Scholar, T. A. F. C. K. C. S. H. K. M. T. et Scholar). The that have no of with the of this We to the of and the for the was by and of of and and of of for and of Z. C. H. Z. M. J. G. Y. and G. Z. C. M. M. and Z. C. Z. and S. D. with and

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