The Experts below are selected from a list of 261 Experts worldwide ranked by ideXlab platform

Shigenori Iwai - One of the best experts on this subject based on the ideXlab platform.

  • Plant organellar DNA polymerases bypass Thymine Glycol using two conserved lysine residues.
    The Biochemical journal, 2020
    Co-Authors: Noe Baruch-torres, Shigenori Iwai, Junpei Yamamoto, Víctor Juarez-quintero, Luis G. Brieba
    Abstract:

    Plant organelles cope with endogenous DNA damaging agents, byproducts of respiration and photosynthesis, and exogenous agents like ultraviolet light. Plant organellar DNA polymerases (DNAPs) are not phylogenetically related to yeast and metazoan DNAPs and they harbor three insertions not present in any other DNAPs. Plant organellar DNAPs from Arabidopsis thaliana (AtPolIA and AtPolIB) are translesion synthesis (TLS) DNAPs able to bypass abasic sites, a lesion that poses a strong block to replicative polymerases. Besides abasic sites, reactive oxidative species and ionizing radiation react with Thymine resulting in Thymine Glycol (Tg), a DNA adduct that is also a strong block to replication. Here, we report that AtPolIA and AtPolIB bypass Tg by inserting an adenine opposite the lesion and efficiently extend from a Tg-A base pair. The TLS ability of AtPolIB is mapped to two conserved lysine residues: K593 and K866. Residue K593 is situated in insertion 1 and K866 is in insertion 3. With basis on the location of both insertions on a structural model of AtPolIIB, we hypothesize that the two positively charged residues interact to form a clamp around the primer-template. In contrast with nuclear and bacterial replication, where lesion bypass involves an interplay between TLS and replicative DNA polymerases, we postulate that plant organellar DNAPs evolved to exert replicative and TLS activities.

  • SPR sensorgrams for the interaction of distamycin A with G·C-14 (), Tg·A-14 () and Tg*·A-14 ()
    2011
    Co-Authors: Tatsuhiko Shimizu, Koichiro Manabe, Shinya Yoshikawa, Yusuke Kawasaki, Shigenori Iwai
    Abstract:

    Copyright information:Taken from "Preferential formation of (5,6)-Thymine Glycol for oligodeoxyribonucleotide synthesis and analysis of drug binding to Thymine Glycol-containing DNA"Nucleic Acids Research 2006;34(1):313-321.Published online 9 Jan 2006PMCID:PMC1326250.© The Author 2006. Published by Oxford University Press. All rights reserved The concentrations of distamycin A are 2, 4, 6, 8, 10, 20 and 30 nM

  • CD spectra of distamycin A complexed with T·A-14 (), G·C-14 (), Tg·A-14 (), Tg*·A-14 (), T·C-14 () and Tg*·C-14 ()
    2011
    Co-Authors: Tatsuhiko Shimizu, Koichiro Manabe, Shinya Yoshikawa, Yusuke Kawasaki, Shigenori Iwai
    Abstract:

    Copyright information:Taken from "Preferential formation of (5,6)-Thymine Glycol for oligodeoxyribonucleotide synthesis and analysis of drug binding to Thymine Glycol-containing DNA"Nucleic Acids Research 2006;34(1):313-321.Published online 9 Jan 2006PMCID:PMC1326250.© The Author 2006. Published by Oxford University Press. All rights reserved The distamycin/duplex ratios are 0 (red), 0.5 (orange), 1.0 (yellow), 1.5 (green) and 2.0 (blue)

  • Efficient conversion of Thymine Glycol into the formamide lesion in oligonucleotides
    Tetrahedron Letters, 2009
    Co-Authors: Tatsuya Toga, Junpei Yamamoto, Shigenori Iwai
    Abstract:

    Abstract Oxidation of (5 R ,6 S )-5,6-dihydro-5,6-dihydroxythymidine (thymidine Glycol) with sodium periodate efficiently produced N -(2-deoxy-β- d - erythro -pentofuranosyl)formamide, a hydroxyl radical-induced decomposition product of pyrimidine bases in DNA, and this method was successfully applied to the conversion of Thymine Glycol in oligonucleotides into the formamide lesion.

  • Human DNA polymerase N (POLN) is a low fidelity enzyme capable of error-free bypass of 5S-Thymine Glycol.
    The Journal of biological chemistry, 2006
    Co-Authors: Kei Ichi Takata, Shigenori Iwai, Tatsuhiko Shimizu, Richard D. Wood
    Abstract:

    Human DNA polymerase N (POLN or pol nu) is the most recently discovered nuclear DNA polymerase in the human genome. It is an A-family DNA polymerase related to Escherichia coli pol I, human POLQ, and Drosophila Mus308. We report the first purification of the recombinant enzyme and examination of its biochemical properties, as a step toward understanding the functions of POLN. Unusual for an A-family DNA polymerase, POLN is a low fidelity enzyme incorporating T opposite template G with a frequency of 0.45 and G opposite template T with a frequency of 0.021. The frequency of misincorporation of T opposite template G is higher than any other known DNA polymerase. POLN has a processivity of DNA synthesis (1-100 nucleotides) similar to the exonuclease-deficient Klenow fragment of E. coli pol I, is inhibited by dideoxynucleotides, and resistant to aphidicolin. The strand displacement activity of POLN was higher than exonuclease-deficient Klenow fragment. Furthermore, POLN can perform translesion synthesis past Thymine Glycol, a common endogenous and radiation-induced product of reactive oxygen species damage to DNA. Thymine Glycol blocks DNA synthesis by most DNA polymerases, but POLN was particularly adept at efficient and accurate translesion synthesis past a 5S-Thymine Glycol.

Sylvie Doublie - One of the best experts on this subject based on the ideXlab platform.

  • structural characterization of viral ortholog of human dna glycosylase neil1 bound to Thymine Glycol or 5 hydroxyuracil containing dna
    Journal of Biological Chemistry, 2012
    Co-Authors: Kayo Imamura, Susan S. Wallace, April M Averill, Sylvie Doublie
    Abstract:

    Thymine Glycol (Tg) and 5-hydroxyuracil (5-OHU) are common oxidized products of pyrimidines, which are recognized and cleaved by two DNA glycosylases of the base excision repair pathway, endonuclease III (Nth) and endonuclease VIII (Nei). Although there are several structures of Nei enzymes unliganded or bound to an abasic (apurinic or apyrimidinic) site, until now there was no structure of an Nei bound to a DNA lesion. Mimivirus Nei1 (MvNei1) is an ortholog of human NEIL1, which was previously crystallized bound to DNA containing an apurinic site (Imamura, K., Wallace, S. S., and Doublie, S. (2009) J. Biol. Chem. 284, 26174–26183). Here, we present two crystal structures of MvNei1 bound to two oxidized pyrimidines, Tg and 5-OHU. Both lesions are flipped out from the DNA helix. Tg is in the anti conformation, whereas 5-OHU adopts both anti and syn conformations in the glycosylase active site. Only two protein side chains (Glu-6 and Tyr-253) are within hydrogen-bonding contact with either damaged base, and mutating these residues did not markedly affect the glycosylase activity. This finding suggests that lesion recognition by Nei occurs before the damaged base flips into the glycosylase active site.

  • Structural Characterization of Viral Ortholog of Human DNA Glycosylase NEIL1 Bound to Thymine Glycol or
    2012
    Co-Authors: Kayo Imamura, Susan S. Wallace, April M Averill, Sylvie Doublie
    Abstract:

    Background: Nei is a DNA glycosylase of the base excision repair pathway. Results: We present two crystal structures of an Nei bound to Thymine Glycol or 5-hydroxyuracil. Conclusion: Mutational analysis of active site residues suggests that lesion recognition happens before the damaged base is everted into the active site. Significance: These are the first structures of any Nei in complex with a damaged base. Thymine Glycol (Tg) and 5-hydroxyuracil (5-OHU) are common oxidized products of pyrimidines, which are recognized and cleaved by two DNA glycosylases of the base excision repair pathway, endonuclease III (Nth) and endonuclease VIII (Nei). Although there are several structures of Nei enzymes unliganded or bound to an abasic (apurinic or apyrimidinic) site, until now there was no structure of an Nei bound to a DNA lesion. Mimivirus Nei1 (MvNei1) is an ortholog of human NEIL1, which was previously crystallized bound to DNA containing an apurinic site (Imamura, K., Wallace, S. S., and Doublie, S. (2009) J. Biol. Chem. 284, 26174 –26183). Here, we present two crystal structures of MvNei1 bound to two oxidized pyrimidines, Tg and 5-OHU. Both lesions are flipped out from the DNA helix. Tg is in the anti conformation, whereas 5-OHU adopts both anti and syn conformations in the glycosylase active site. Only two protein side chains (Glu-6 and Tyr-253) are within hydrogen

  • a crystallographic study of the role of sequence context in Thymine Glycol bypass by a replicative dna polymerase serendipitously sheds light on the exonuclease complex
    Journal of Molecular Biology, 2011
    Co-Authors: Pierre Aller, Susan S. Wallace, Stéphanie Duclos, Sylvie Doublie
    Abstract:

    Thymine Glycol (Tg) is the most common oxidation product of Thymine and is known to be a strong block for replicative DNA polymerases. A previously solved structure of the bacteriophage RB69 DNA polymerase (RB69 gp43) in complex with Tg in the sequence context 5’-G-Tg-G shed light on how Tg blocks primer elongation: The protruding methyl group of the oxidized Thymine displaces the adjacent 5’-G which can no longer serve as a template for primer elongation. [Aller, P., Rould, M.A., Hogg, M, Wallace, S.S., & Doublie S. (2007) PNAS 104, 814–818] Several studies showed that in the 5’-C-Tg-Purine sequence context Tg is more likely to be bypassed by Klenow fragment, a family A DNA polymerase. We set out to investigate the role of sequence context on Tg bypass in a B family polymerase and solved the crystal structures of the bacteriophage RB69 DNA polymerase in complex with Tg containing DNA in the three remaining sequence contexts: 5’-N-Tg-G with N=A, T, or C. A combination of several factors influence Tg bypass, including the associated exonuclease activity, the nature of the 3’and 5’ bases surrounding Tg and the cis/trans interconversion of Tg. We also visualized for the first time the structure of a well-ordered exonuclease complex, allowing us to identify and confirm the role of key residues (Phe123, Met256, and Tyr257) in strand separation and the stabilization of the primer strand in the exonuclease site.

  • A Crystallographic Study of the Role of Sequence Context in Thymine Glycol Bypass by a Replicative DNA Polymerase Serendipitously Sheds Light on the Exonuclease Complex
    Journal of molecular biology, 2011
    Co-Authors: Pierre Aller, Susan S. Wallace, Stéphanie Duclos, Sylvie Doublie
    Abstract:

    Thymine Glycol (Tg) is the most common oxidation product of Thymine and is known to be a strong block to replicative DNA polymerases. A previously solved structure of the bacteriophage RB69 DNA polymerase (RB69 gp43) in complex with Tg in the sequence context 5'-G-Tg-G shed light on how Tg blocks primer elongation: The protruding methyl group of the oxidized Thymine displaces the adjacent 5'-G, which can no longer serve as a template for primer elongation [Aller, P., Rould, M. A., Hogg, M, Wallace, S. S. & Doublié S. (2007). A structural rationale for stalling of a replicative DNA polymerase at the most common oxidative Thymine lesion, Thymine Glycol. Proc. Natl. Acad. Sci. USA, 104, 814-818.]. Several studies showed that in the sequence context 5'-C-Tg-purine, Tg is more likely to be bypassed by Klenow fragment, an A-family DNA polymerase. We set out to investigate the role of sequence context in Tg bypass in a B-family polymerase and to solve the crystal structures of the bacteriophage RB69 DNA polymerase in complex with Tg-containing DNA in the three remaining sequence contexts: 5'-A-Tg-G, 5'-T-Tg-G, and 5'-C-Tg-G. A combination of several factors-including the associated exonuclease activity, the nature of the 3' and 5' bases surrounding Tg, and the cis-trans interconversion of Tg-influences Tg bypass. We also visualized for the first time the structure of a well-ordered exonuclease complex, allowing us to identify and confirm the role of key residues (Phe123, Met256, and Tyr257) in strand separation and in the stabilization of the primer strand in the exonuclease site.

  • A structural rationale for stalling of a replicative DNA polymerase at the most common oxidative Thymine lesion, Thymine Glycol.
    Proceedings of the National Academy of Sciences of the United States of America, 2007
    Co-Authors: Pierre Aller, Susan S. Wallace, Mark A. Rould, Matthew Hogg, Sylvie Doublie
    Abstract:

    Thymine Glycol (Tg) is a common product of oxidation and ionizing radiation, including that used for cancer treatment. Although Tg is a poor mutagenic lesion, it has been shown to present a strong block to both repair and replicative DNA polymerases. The 2.65-A crystal structure of a binary complex of the replicative RB69 DNA polymerase with DNA shows that the templating Tg is intrahelical and forms a regular Watson-Crick base pair with the incorporated A. The C5 methyl group protrudes axially from the ring of the damaged pyrimidine and hinders stacking of the adjacent 5' template guanine. The position of the displaced 5' template guanine is such that the next incoming nucleotide cannot be incorporated into the growing primer strand, and it explains why primer extension past the lesion is prohibited even though DNA polymerases can readily incorporate an A across from the Tg lesion.

Michael P Stone - One of the best experts on this subject based on the ideXlab platform.

  • binding of the human nucleotide excision repair proteins xpa and xpc hr23b to the 5r Thymine Glycol lesion and structure of the cis 5r 6s Thymine Glycol epimer in the 5 gtgg 3 sequence destabilization of two base pairs at the lesion site
    Nucleic Acids Research, 2010
    Co-Authors: Kyle L Brown, Ashis K. Basu, Marina Roginskaya, Yue Zou, Alvin Altamirano, Michael P Stone
    Abstract:

    The 5R Thymine Glycol (5R-Tg) DNA lesion exists as a mixture of cis-(5R,6S) and trans-(5R,6R) epimers; these modulate base excision repair. We examine the 7:3 cis-(5R,6S):trans-(5R,6R) mixture of epimers paired opposite adenine in the 5'-GTgG-3' sequence with regard to nucleotide excision repair. Human XPA recognizes the lesion comparably to the C8-dG acetylaminoflourene (AAF) adduct, whereas XPC/HR23B recognition of Tg is superior. 5R-Tg is processed by the Escherichia coli UvrA and UvrABC proteins less efficiently than the C8-dG AAF adduct. For the cis-(5R, 6S) epimer Tg and A are inserted into the helix, remaining in the Watson-Crick alignment. The Tg N3H imine and A N(6) amine protons undergo increased solvent exchange. Stacking between Tg and the 3'-neighbor G*C base pair is disrupted. The solvent accessible surface and T(2) relaxation of Tg increases. Molecular dynamics calculations predict that the axial conformation of the Tg CH(3) group is favored; propeller twisting of the Tg*A pair and hydrogen bonding between Tg OH6 and the N7 atom of the 3'-neighbor guanine alleviate steric clash with the 5'-neighbor base pair. Tg also destabilizes the 5'-neighbor G*C base pair. This may facilitate flipping both base pairs from the helix, enabling XPC/HR23B recognition prior to recruitment of XPA.

  • Structural Consequences of Epimerization of Thymine Glycol Lesions in Duplex DNA: Implications for DNA Repair
    2010
    Co-Authors: Kyle L Brown, Ashis K. Basu, Marina Roginskaya, Yue Zou, Alvin Altamirano, Michael P Stone
    Abstract:

    Thymine Glycol (Tg), 5,6-dihydroxy-5,6-dihydroThymine, forms in DNA by reaction of Thymine with reactive oxygen species. It exists as two diastereomeric pairs of epimers, the 5R cis, trans pair (5R,6S;5R,6R) and the 5S cis, trans pair (5S,6R; 5S,6S). The 5R pair is more abundant. At 30 °C, a 70%:30% cis:trans ratio of epimers is present in this sequence when SR-Tg is opposite dA. For the cis epimer Tg and A remain in the Watson-Crick alignment. The Tg N3H imine and A N 6 amine protons undergo increased solvent exchange. Stacking between Tg and the 3'-neighbor G•C base pair is disrupted. The solvent accessible surface and T 2 relaxation of Tg increases. Molecular dynamics calculations predict that the axial conformation of the Tg CH 3 group is favored; propeller twisting of the Tg•A pair and hydrogen bonding between Tg OH6 and the N7 atom of the 3'-neighbor guanine alleviate steric clash with the 5'-neighbor base pair. Tg also destabilizes the 5'-neighbor G•C base pair. Under these conditions, the human NER protein XPA binds to the 5R-Tg lesion comparably to the C8-dG acetylaminoflourene (AAF) adduct, whereas XPC/HR23B binding of the Tg lesion is superior than to the AAF adduct. In comparison, this lesion is processed by the Escherichia coli UvrA and UvrABC proteins less efficiently than the C8-dG AAF adduct. The destabilization of two base pairs by the cis epimer may facilitate flipping both base pairs from the helix, enabling XPC/HR23B binding prior to recruitment of XPA. When 5R-Tg pairs opposite dG in this sequence context only the cis epimer is observed. Tg assumes the wobble orientation and stacks below the 5'-neighbor dG, while the mismatched dG stacks below the 5'-neighbor dC. Stacking between Tg and the 3'-neighbor G•C base pair is disrupted. Differences in base excision repair of the Tg•G and Tg•A pairs by hNEIL1 may be related to the wobble orientation of the cis Tg epimer in the Tg•G pair, and the lack of hydrogen bonding between the Tg OH groups and the N7 atom of the 3'-neighbor dG. Hydrogen bonding between Tg 6 OH6→G 7 N7 in the Tg•A pair may increase the energetic barrier with regard to flipping of the Tg lesion into the active site pocket of the glycosylase, hindering repair.

  • Binding of the human nucleotide excision repair proteins XPA and XPC/HR23B to the 5R-Thymine Glycol lesion and structure of the cis-(5R,6S) Thymine Glycol epimer in the 5′-GTgG-3′ sequence: destabilization of two base pairs at the lesion site
    Nucleic acids research, 2009
    Co-Authors: Kyle L Brown, Ashis K. Basu, Marina Roginskaya, Yue Zou, Alvin Altamirano, Michael P Stone
    Abstract:

    The 5R Thymine Glycol (5R-Tg) DNA lesion exists as a mixture of cis-(5R,6S) and trans-(5R,6R) epimers; these modulate base excision repair. We examine the 7:3 cis-(5R,6S):trans-(5R,6R) mixture of epimers paired opposite adenine in the 5'-GTgG-3' sequence with regard to nucleotide excision repair. Human XPA recognizes the lesion comparably to the C8-dG acetylaminoflourene (AAF) adduct, whereas XPC/HR23B recognition of Tg is superior. 5R-Tg is processed by the Escherichia coli UvrA and UvrABC proteins less efficiently than the C8-dG AAF adduct. For the cis-(5R, 6S) epimer Tg and A are inserted into the helix, remaining in the Watson-Crick alignment. The Tg N3H imine and A N(6) amine protons undergo increased solvent exchange. Stacking between Tg and the 3'-neighbor G*C base pair is disrupted. The solvent accessible surface and T(2) relaxation of Tg increases. Molecular dynamics calculations predict that the axial conformation of the Tg CH(3) group is favored; propeller twisting of the Tg*A pair and hydrogen bonding between Tg OH6 and the N7 atom of the 3'-neighbor guanine alleviate steric clash with the 5'-neighbor base pair. Tg also destabilizes the 5'-neighbor G*C base pair. This may facilitate flipping both base pairs from the helix, enabling XPC/HR23B recognition prior to recruitment of XPA.

  • the cis 5r 6s Thymine Glycol lesion occupies the wobble position when mismatched with deoxyguanosine in dna
    Biochemistry, 2009
    Co-Authors: Kyle L Brown, Ashis K. Basu, Michael P Stone
    Abstract:

    Oxidative damage to 5-methylcytosine in DNA, followed by deamination, yields Thymine Glycol (Tg), 5,6-dihydroxy-5,6-dihydroThymine, mispaired with deoxyguanosine. The structure of the 5R Tg·G mismatch pair has been refined using a combination of simulated annealing and isothermal molecular dynamics calculations restrained by NMR-derived distance restraints and torsion angle restraints in 5′-d(G1T2G3C4G5Tg6G7T8T9T10G11T12)-3′·5′-d(A13C14A15A16A17C18G19C20G21C22A23C24)-3′; Tg = 5R Tg. In this duplex the cis-5R,6S:trans-5R,6R equilibrium favors the cis-5R,6S epimer [Brown, K. L., Adams, T., Jasti, V. P., Basu, A. K., and Stone, M. P. (2008) J. Am. Chem. Soc. 130, 11701−11710]. The cis-5R,6S Tg lesion is in the wobble orientation such that Tg6 O2 is proximate to G19 N1H and Tg6 N3H is proximate to G19 O6. Both Tg6 and the mismatched nucleotide G19 remain stacked in the helix. The Tg6 nucleotide shifts toward the major groove and stacks below the 5′-neighbor base G5, while its complement G19 stacks below the 5...

  • The cis-(5R,6S)-Thymine Glycol lesion occupies the wobble position when mismatched with deoxyguanosine in DNA.
    Biochemistry, 2009
    Co-Authors: Kyle L Brown, Ashis K. Basu, Michael P Stone
    Abstract:

    Oxidative damage to 5-methylcytosine in DNA, followed by deamination, yields Thymine Glycol (Tg), 5,6-dihydroxy-5,6-dihydroThymine, mispaired with deoxyguanosine. The structure of the 5R Tg.G mismatch pair has been refined using a combination of simulated annealing and isothermal molecular dynamics calculations restrained by NMR-derived distance restraints and torsion angle restraints in 5'-d(G(1)T(2)G(3)C(4)G(5)Tg(6)G(7)T(8)T(9)T(10)G(11)T(12))-3'.5'-d(A(13)C(14)A(15)A(16)A(17)C(18)G(19)C(20)G(21)C(22)A(23)C(24))-3'; Tg = 5R Tg. In this duplex the cis-5R,6S:trans-5R,6R equilibrium favors the cis-5R,6S epimer [Brown, K. L., Adams, T., Jasti, V. P., Basu, A. K., and Stone, M. P. (2008) J. Am. Chem. Soc. 130, 11701-11710]. The cis-5R,6S Tg lesion is in the wobble orientation such that Tg(6) O(2) is proximate to G(19) N1H and Tg(6) N3H is proximate to G(19) O(6). Both Tg(6) and the mismatched nucleotide G(19) remain stacked in the helix. The Tg(6) nucleotide shifts toward the major groove and stacks below the 5'-neighbor base G(5), while its complement G(19) stacks below the 5'-neighbor C(20). In the 3'-direction, stacking between Tg(6) and the G(7).C(18) base pair is disrupted. The solvent-accessible surface area of the Tg nucleotide increases as compared to the native Watson-Crick hydrogen-bonded T.A base pair. An increase in T(2) relaxation rates for the Tg(6) base protons is attributed to puckering of the Tg base, accompanied by increased disorder at the Tg.G mismatch pair. The axial vs equatorial conformation of the Tg(6) CH(3) group cannot be determined with certainty from the NMR data. The rMD trajectories suggest that in either the axial or equatorial conformations the cis-5R,6S Tg lesion does not form strong intrastrand hydrogen bonds with the imidazole N7 atom of the 3'-neighbor purine G(7). The wobble pairing and disorder of the Tg.G mismatch correlate with the reduced thermodynamic stability of the mismatch and likely modulate its recognition by DNA base excision repair systems.

Susan S. Wallace - One of the best experts on this subject based on the ideXlab platform.

  • structural characterization of viral ortholog of human dna glycosylase neil1 bound to Thymine Glycol or 5 hydroxyuracil containing dna
    Journal of Biological Chemistry, 2012
    Co-Authors: Kayo Imamura, Susan S. Wallace, April M Averill, Sylvie Doublie
    Abstract:

    Thymine Glycol (Tg) and 5-hydroxyuracil (5-OHU) are common oxidized products of pyrimidines, which are recognized and cleaved by two DNA glycosylases of the base excision repair pathway, endonuclease III (Nth) and endonuclease VIII (Nei). Although there are several structures of Nei enzymes unliganded or bound to an abasic (apurinic or apyrimidinic) site, until now there was no structure of an Nei bound to a DNA lesion. Mimivirus Nei1 (MvNei1) is an ortholog of human NEIL1, which was previously crystallized bound to DNA containing an apurinic site (Imamura, K., Wallace, S. S., and Doublie, S. (2009) J. Biol. Chem. 284, 26174–26183). Here, we present two crystal structures of MvNei1 bound to two oxidized pyrimidines, Tg and 5-OHU. Both lesions are flipped out from the DNA helix. Tg is in the anti conformation, whereas 5-OHU adopts both anti and syn conformations in the glycosylase active site. Only two protein side chains (Glu-6 and Tyr-253) are within hydrogen-bonding contact with either damaged base, and mutating these residues did not markedly affect the glycosylase activity. This finding suggests that lesion recognition by Nei occurs before the damaged base flips into the glycosylase active site.

  • Structural Characterization of Viral Ortholog of Human DNA Glycosylase NEIL1 Bound to Thymine Glycol or
    2012
    Co-Authors: Kayo Imamura, Susan S. Wallace, April M Averill, Sylvie Doublie
    Abstract:

    Background: Nei is a DNA glycosylase of the base excision repair pathway. Results: We present two crystal structures of an Nei bound to Thymine Glycol or 5-hydroxyuracil. Conclusion: Mutational analysis of active site residues suggests that lesion recognition happens before the damaged base is everted into the active site. Significance: These are the first structures of any Nei in complex with a damaged base. Thymine Glycol (Tg) and 5-hydroxyuracil (5-OHU) are common oxidized products of pyrimidines, which are recognized and cleaved by two DNA glycosylases of the base excision repair pathway, endonuclease III (Nth) and endonuclease VIII (Nei). Although there are several structures of Nei enzymes unliganded or bound to an abasic (apurinic or apyrimidinic) site, until now there was no structure of an Nei bound to a DNA lesion. Mimivirus Nei1 (MvNei1) is an ortholog of human NEIL1, which was previously crystallized bound to DNA containing an apurinic site (Imamura, K., Wallace, S. S., and Doublie, S. (2009) J. Biol. Chem. 284, 26174 –26183). Here, we present two crystal structures of MvNei1 bound to two oxidized pyrimidines, Tg and 5-OHU. Both lesions are flipped out from the DNA helix. Tg is in the anti conformation, whereas 5-OHU adopts both anti and syn conformations in the glycosylase active site. Only two protein side chains (Glu-6 and Tyr-253) are within hydrogen

  • a crystallographic study of the role of sequence context in Thymine Glycol bypass by a replicative dna polymerase serendipitously sheds light on the exonuclease complex
    Journal of Molecular Biology, 2011
    Co-Authors: Pierre Aller, Susan S. Wallace, Stéphanie Duclos, Sylvie Doublie
    Abstract:

    Thymine Glycol (Tg) is the most common oxidation product of Thymine and is known to be a strong block for replicative DNA polymerases. A previously solved structure of the bacteriophage RB69 DNA polymerase (RB69 gp43) in complex with Tg in the sequence context 5’-G-Tg-G shed light on how Tg blocks primer elongation: The protruding methyl group of the oxidized Thymine displaces the adjacent 5’-G which can no longer serve as a template for primer elongation. [Aller, P., Rould, M.A., Hogg, M, Wallace, S.S., & Doublie S. (2007) PNAS 104, 814–818] Several studies showed that in the 5’-C-Tg-Purine sequence context Tg is more likely to be bypassed by Klenow fragment, a family A DNA polymerase. We set out to investigate the role of sequence context on Tg bypass in a B family polymerase and solved the crystal structures of the bacteriophage RB69 DNA polymerase in complex with Tg containing DNA in the three remaining sequence contexts: 5’-N-Tg-G with N=A, T, or C. A combination of several factors influence Tg bypass, including the associated exonuclease activity, the nature of the 3’and 5’ bases surrounding Tg and the cis/trans interconversion of Tg. We also visualized for the first time the structure of a well-ordered exonuclease complex, allowing us to identify and confirm the role of key residues (Phe123, Met256, and Tyr257) in strand separation and the stabilization of the primer strand in the exonuclease site.

  • A Crystallographic Study of the Role of Sequence Context in Thymine Glycol Bypass by a Replicative DNA Polymerase Serendipitously Sheds Light on the Exonuclease Complex
    Journal of molecular biology, 2011
    Co-Authors: Pierre Aller, Susan S. Wallace, Stéphanie Duclos, Sylvie Doublie
    Abstract:

    Thymine Glycol (Tg) is the most common oxidation product of Thymine and is known to be a strong block to replicative DNA polymerases. A previously solved structure of the bacteriophage RB69 DNA polymerase (RB69 gp43) in complex with Tg in the sequence context 5'-G-Tg-G shed light on how Tg blocks primer elongation: The protruding methyl group of the oxidized Thymine displaces the adjacent 5'-G, which can no longer serve as a template for primer elongation [Aller, P., Rould, M. A., Hogg, M, Wallace, S. S. & Doublié S. (2007). A structural rationale for stalling of a replicative DNA polymerase at the most common oxidative Thymine lesion, Thymine Glycol. Proc. Natl. Acad. Sci. USA, 104, 814-818.]. Several studies showed that in the sequence context 5'-C-Tg-purine, Tg is more likely to be bypassed by Klenow fragment, an A-family DNA polymerase. We set out to investigate the role of sequence context in Tg bypass in a B-family polymerase and to solve the crystal structures of the bacteriophage RB69 DNA polymerase in complex with Tg-containing DNA in the three remaining sequence contexts: 5'-A-Tg-G, 5'-T-Tg-G, and 5'-C-Tg-G. A combination of several factors-including the associated exonuclease activity, the nature of the 3' and 5' bases surrounding Tg, and the cis-trans interconversion of Tg-influences Tg bypass. We also visualized for the first time the structure of a well-ordered exonuclease complex, allowing us to identify and confirm the role of key residues (Phe123, Met256, and Tyr257) in strand separation and in the stabilization of the primer strand in the exonuclease site.

  • A structural rationale for stalling of a replicative DNA polymerase at the most common oxidative Thymine lesion, Thymine Glycol.
    Proceedings of the National Academy of Sciences of the United States of America, 2007
    Co-Authors: Pierre Aller, Susan S. Wallace, Mark A. Rould, Matthew Hogg, Sylvie Doublie
    Abstract:

    Thymine Glycol (Tg) is a common product of oxidation and ionizing radiation, including that used for cancer treatment. Although Tg is a poor mutagenic lesion, it has been shown to present a strong block to both repair and replicative DNA polymerases. The 2.65-A crystal structure of a binary complex of the replicative RB69 DNA polymerase with DNA shows that the templating Tg is intrahelical and forms a regular Watson-Crick base pair with the incorporated A. The C5 methyl group protrudes axially from the ring of the damaged pyrimidine and hinders stacking of the adjacent 5' template guanine. The position of the displaced 5' template guanine is such that the next incoming nucleotide cannot be incorporated into the growing primer strand, and it explains why primer extension past the lesion is prohibited even though DNA polymerases can readily incorporate an A across from the Tg lesion.

Ashis K. Basu - One of the best experts on this subject based on the ideXlab platform.

  • Structural Consequences of Epimerization of Thymine Glycol Lesions in Duplex DNA: Implications for DNA Repair
    2010
    Co-Authors: Kyle L Brown, Ashis K. Basu, Marina Roginskaya, Yue Zou, Alvin Altamirano, Michael P Stone
    Abstract:

    Thymine Glycol (Tg), 5,6-dihydroxy-5,6-dihydroThymine, forms in DNA by reaction of Thymine with reactive oxygen species. It exists as two diastereomeric pairs of epimers, the 5R cis, trans pair (5R,6S;5R,6R) and the 5S cis, trans pair (5S,6R; 5S,6S). The 5R pair is more abundant. At 30 °C, a 70%:30% cis:trans ratio of epimers is present in this sequence when SR-Tg is opposite dA. For the cis epimer Tg and A remain in the Watson-Crick alignment. The Tg N3H imine and A N 6 amine protons undergo increased solvent exchange. Stacking between Tg and the 3'-neighbor G•C base pair is disrupted. The solvent accessible surface and T 2 relaxation of Tg increases. Molecular dynamics calculations predict that the axial conformation of the Tg CH 3 group is favored; propeller twisting of the Tg•A pair and hydrogen bonding between Tg OH6 and the N7 atom of the 3'-neighbor guanine alleviate steric clash with the 5'-neighbor base pair. Tg also destabilizes the 5'-neighbor G•C base pair. Under these conditions, the human NER protein XPA binds to the 5R-Tg lesion comparably to the C8-dG acetylaminoflourene (AAF) adduct, whereas XPC/HR23B binding of the Tg lesion is superior than to the AAF adduct. In comparison, this lesion is processed by the Escherichia coli UvrA and UvrABC proteins less efficiently than the C8-dG AAF adduct. The destabilization of two base pairs by the cis epimer may facilitate flipping both base pairs from the helix, enabling XPC/HR23B binding prior to recruitment of XPA. When 5R-Tg pairs opposite dG in this sequence context only the cis epimer is observed. Tg assumes the wobble orientation and stacks below the 5'-neighbor dG, while the mismatched dG stacks below the 5'-neighbor dC. Stacking between Tg and the 3'-neighbor G•C base pair is disrupted. Differences in base excision repair of the Tg•G and Tg•A pairs by hNEIL1 may be related to the wobble orientation of the cis Tg epimer in the Tg•G pair, and the lack of hydrogen bonding between the Tg OH groups and the N7 atom of the 3'-neighbor dG. Hydrogen bonding between Tg 6 OH6→G 7 N7 in the Tg•A pair may increase the energetic barrier with regard to flipping of the Tg lesion into the active site pocket of the glycosylase, hindering repair.

  • binding of the human nucleotide excision repair proteins xpa and xpc hr23b to the 5r Thymine Glycol lesion and structure of the cis 5r 6s Thymine Glycol epimer in the 5 gtgg 3 sequence destabilization of two base pairs at the lesion site
    Nucleic Acids Research, 2010
    Co-Authors: Kyle L Brown, Ashis K. Basu, Marina Roginskaya, Yue Zou, Alvin Altamirano, Michael P Stone
    Abstract:

    The 5R Thymine Glycol (5R-Tg) DNA lesion exists as a mixture of cis-(5R,6S) and trans-(5R,6R) epimers; these modulate base excision repair. We examine the 7:3 cis-(5R,6S):trans-(5R,6R) mixture of epimers paired opposite adenine in the 5'-GTgG-3' sequence with regard to nucleotide excision repair. Human XPA recognizes the lesion comparably to the C8-dG acetylaminoflourene (AAF) adduct, whereas XPC/HR23B recognition of Tg is superior. 5R-Tg is processed by the Escherichia coli UvrA and UvrABC proteins less efficiently than the C8-dG AAF adduct. For the cis-(5R, 6S) epimer Tg and A are inserted into the helix, remaining in the Watson-Crick alignment. The Tg N3H imine and A N(6) amine protons undergo increased solvent exchange. Stacking between Tg and the 3'-neighbor G*C base pair is disrupted. The solvent accessible surface and T(2) relaxation of Tg increases. Molecular dynamics calculations predict that the axial conformation of the Tg CH(3) group is favored; propeller twisting of the Tg*A pair and hydrogen bonding between Tg OH6 and the N7 atom of the 3'-neighbor guanine alleviate steric clash with the 5'-neighbor base pair. Tg also destabilizes the 5'-neighbor G*C base pair. This may facilitate flipping both base pairs from the helix, enabling XPC/HR23B recognition prior to recruitment of XPA.

  • Binding of the human nucleotide excision repair proteins XPA and XPC/HR23B to the 5R-Thymine Glycol lesion and structure of the cis-(5R,6S) Thymine Glycol epimer in the 5′-GTgG-3′ sequence: destabilization of two base pairs at the lesion site
    Nucleic acids research, 2009
    Co-Authors: Kyle L Brown, Ashis K. Basu, Marina Roginskaya, Yue Zou, Alvin Altamirano, Michael P Stone
    Abstract:

    The 5R Thymine Glycol (5R-Tg) DNA lesion exists as a mixture of cis-(5R,6S) and trans-(5R,6R) epimers; these modulate base excision repair. We examine the 7:3 cis-(5R,6S):trans-(5R,6R) mixture of epimers paired opposite adenine in the 5'-GTgG-3' sequence with regard to nucleotide excision repair. Human XPA recognizes the lesion comparably to the C8-dG acetylaminoflourene (AAF) adduct, whereas XPC/HR23B recognition of Tg is superior. 5R-Tg is processed by the Escherichia coli UvrA and UvrABC proteins less efficiently than the C8-dG AAF adduct. For the cis-(5R, 6S) epimer Tg and A are inserted into the helix, remaining in the Watson-Crick alignment. The Tg N3H imine and A N(6) amine protons undergo increased solvent exchange. Stacking between Tg and the 3'-neighbor G*C base pair is disrupted. The solvent accessible surface and T(2) relaxation of Tg increases. Molecular dynamics calculations predict that the axial conformation of the Tg CH(3) group is favored; propeller twisting of the Tg*A pair and hydrogen bonding between Tg OH6 and the N7 atom of the 3'-neighbor guanine alleviate steric clash with the 5'-neighbor base pair. Tg also destabilizes the 5'-neighbor G*C base pair. This may facilitate flipping both base pairs from the helix, enabling XPC/HR23B recognition prior to recruitment of XPA.

  • the cis 5r 6s Thymine Glycol lesion occupies the wobble position when mismatched with deoxyguanosine in dna
    Biochemistry, 2009
    Co-Authors: Kyle L Brown, Ashis K. Basu, Michael P Stone
    Abstract:

    Oxidative damage to 5-methylcytosine in DNA, followed by deamination, yields Thymine Glycol (Tg), 5,6-dihydroxy-5,6-dihydroThymine, mispaired with deoxyguanosine. The structure of the 5R Tg·G mismatch pair has been refined using a combination of simulated annealing and isothermal molecular dynamics calculations restrained by NMR-derived distance restraints and torsion angle restraints in 5′-d(G1T2G3C4G5Tg6G7T8T9T10G11T12)-3′·5′-d(A13C14A15A16A17C18G19C20G21C22A23C24)-3′; Tg = 5R Tg. In this duplex the cis-5R,6S:trans-5R,6R equilibrium favors the cis-5R,6S epimer [Brown, K. L., Adams, T., Jasti, V. P., Basu, A. K., and Stone, M. P. (2008) J. Am. Chem. Soc. 130, 11701−11710]. The cis-5R,6S Tg lesion is in the wobble orientation such that Tg6 O2 is proximate to G19 N1H and Tg6 N3H is proximate to G19 O6. Both Tg6 and the mismatched nucleotide G19 remain stacked in the helix. The Tg6 nucleotide shifts toward the major groove and stacks below the 5′-neighbor base G5, while its complement G19 stacks below the 5...

  • The cis-(5R,6S)-Thymine Glycol lesion occupies the wobble position when mismatched with deoxyguanosine in DNA.
    Biochemistry, 2009
    Co-Authors: Kyle L Brown, Ashis K. Basu, Michael P Stone
    Abstract:

    Oxidative damage to 5-methylcytosine in DNA, followed by deamination, yields Thymine Glycol (Tg), 5,6-dihydroxy-5,6-dihydroThymine, mispaired with deoxyguanosine. The structure of the 5R Tg.G mismatch pair has been refined using a combination of simulated annealing and isothermal molecular dynamics calculations restrained by NMR-derived distance restraints and torsion angle restraints in 5'-d(G(1)T(2)G(3)C(4)G(5)Tg(6)G(7)T(8)T(9)T(10)G(11)T(12))-3'.5'-d(A(13)C(14)A(15)A(16)A(17)C(18)G(19)C(20)G(21)C(22)A(23)C(24))-3'; Tg = 5R Tg. In this duplex the cis-5R,6S:trans-5R,6R equilibrium favors the cis-5R,6S epimer [Brown, K. L., Adams, T., Jasti, V. P., Basu, A. K., and Stone, M. P. (2008) J. Am. Chem. Soc. 130, 11701-11710]. The cis-5R,6S Tg lesion is in the wobble orientation such that Tg(6) O(2) is proximate to G(19) N1H and Tg(6) N3H is proximate to G(19) O(6). Both Tg(6) and the mismatched nucleotide G(19) remain stacked in the helix. The Tg(6) nucleotide shifts toward the major groove and stacks below the 5'-neighbor base G(5), while its complement G(19) stacks below the 5'-neighbor C(20). In the 3'-direction, stacking between Tg(6) and the G(7).C(18) base pair is disrupted. The solvent-accessible surface area of the Tg nucleotide increases as compared to the native Watson-Crick hydrogen-bonded T.A base pair. An increase in T(2) relaxation rates for the Tg(6) base protons is attributed to puckering of the Tg base, accompanied by increased disorder at the Tg.G mismatch pair. The axial vs equatorial conformation of the Tg(6) CH(3) group cannot be determined with certainty from the NMR data. The rMD trajectories suggest that in either the axial or equatorial conformations the cis-5R,6S Tg lesion does not form strong intrastrand hydrogen bonds with the imidazole N7 atom of the 3'-neighbor purine G(7). The wobble pairing and disorder of the Tg.G mismatch correlate with the reduced thermodynamic stability of the mismatch and likely modulate its recognition by DNA base excision repair systems.