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. 1998 Sep 1;17(17):5214–5226. doi: 10.1093/emboj/17.17.5214

Base excision repair initiation revealed by crystal structures and binding kinetics of human uracil-DNA glycosylase with DNA.

S S Parikh 1, C D Mol 1, G Slupphaug 1, S Bharati 1, H E Krokan 1, J A Tainer 1
PMCID: PMC1170849  PMID: 9724657

Abstract

Three high-resolution crystal structures of DNA complexes with wild-type and mutant human uracil-DNA glycosylase (UDG), coupled kinetic characterizations and comparisons with the refined unbound UDG structure help resolve fundamental issues in the initiation of DNA base excision repair (BER): damage detection, nucleotide flipping versus extrahelical nucleotide capture, avoidance of apurinic/apyrimidinic (AP) site toxicity and coupling of damage-specific and damage-general BER steps. Structural and kinetic results suggest that UDG binds, kinks and compresses the DNA backbone with a 'Ser-Pro pinch' and scans the minor groove for damage. Concerted shifts in UDG simultaneously form the catalytically competent active site and induce further compression and kinking of the double-stranded DNA backbone only at uracil and AP sites, where these nucleotides can flip at the phosphate-sugar junction into a complementary specificity pocket. Unexpectedly, UDG binds to AP sites more tightly and more rapidly than to uracil-containing DNA, and thus may protect cells sterically from AP site toxicity. Furthermore, AP-endonuclease, which catalyzes the first damage-general step of BER, enhances UDG activity, most likely by inducing UDG release via shared minor groove contacts and flipped AP site binding. Thus, AP site binding may couple damage-specific and damage-general steps of BER without requiring direct protein-protein interactions.

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Selected References

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  1. Barrett T. E., Savva R., Panayotou G., Barlow T., Brown T., Jiricny J., Pearl L. H. Crystal structure of a G:T/U mismatch-specific DNA glycosylase: mismatch recognition by complementary-strand interactions. Cell. 1998 Jan 9;92(1):117–129. doi: 10.1016/s0092-8674(00)80904-6. [DOI] [PubMed] [Google Scholar]
  2. Bennett R. A., Wilson D. M., 3rd, Wong D., Demple B. Interaction of human apurinic endonuclease and DNA polymerase beta in the base excision repair pathway. Proc Natl Acad Sci U S A. 1997 Jul 8;94(14):7166–7169. doi: 10.1073/pnas.94.14.7166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brünger A. T., Kuriyan J., Karplus M. Crystallographic R factor refinement by molecular dynamics. Science. 1987 Jan 23;235(4787):458–460. doi: 10.1126/science.235.4787.458. [DOI] [PubMed] [Google Scholar]
  4. Caldecott K. W., Tucker J. D., Stanker L. H., Thompson L. H. Characterization of the XRCC1-DNA ligase III complex in vitro and its absence from mutant hamster cells. Nucleic Acids Res. 1995 Dec 11;23(23):4836–4843. doi: 10.1093/nar/23.23.4836. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chee M. S., Bankier A. T., Beck S., Bohni R., Brown C. M., Cerny R., Horsnell T., Hutchison C. A., 3rd, Kouzarides T., Martignetti J. A. Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD169. Curr Top Microbiol Immunol. 1990;154:125–169. doi: 10.1007/978-3-642-74980-3_6. [DOI] [PubMed] [Google Scholar]
  6. Cheng X., Blumenthal R. M. Finding a basis for flipping bases. Structure. 1996 Jun 15;4(6):639–645. doi: 10.1016/s0969-2126(96)00068-8. [DOI] [PubMed] [Google Scholar]
  7. Domena J. D., Timmer R. T., Dicharry S. A., Mosbaugh D. W. Purification and properties of mitochondrial uracil-DNA glycosylase from rat liver. Biochemistry. 1988 Sep 6;27(18):6742–6751. doi: 10.1021/bi00418a015. [DOI] [PubMed] [Google Scholar]
  8. Goebel S. J., Johnson G. P., Perkus M. E., Davis S. W., Winslow J. P., Paoletti E. The complete DNA sequence of vaccinia virus. Virology. 1990 Nov;179(1):247-66, 517-63. doi: 10.1016/0042-6822(90)90294-2. [DOI] [PubMed] [Google Scholar]
  9. Gorman M. A., Morera S., Rothwell D. G., de La Fortelle E., Mol C. D., Tainer J. A., Hickson I. D., Freemont P. S. The crystal structure of the human DNA repair endonuclease HAP1 suggests the recognition of extra-helical deoxyribose at DNA abasic sites. EMBO J. 1997 Nov 3;16(21):6548–6558. doi: 10.1093/emboj/16.21.6548. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Haug T., Skorpen F., Lund H., Krokan H. E. Structure of the gene for human uracil-DNA glycosylase and analysis of the promoter function. FEBS Lett. 1994 Oct 17;353(2):180–184. doi: 10.1016/0014-5793(94)01042-0. [DOI] [PubMed] [Google Scholar]
  11. Kavli B., Slupphaug G., Mol C. D., Arvai A. S., Peterson S. B., Tainer J. A., Krokan H. E. Excision of cytosine and thymine from DNA by mutants of human uracil-DNA glycosylase. EMBO J. 1996 Jul 1;15(13):3442–3447. [PMC free article] [PubMed] [Google Scholar]
  12. Kingma P. S., Osheroff N. Apurinic sites are position-specific topoisomerase II poisons. J Biol Chem. 1997 Jan 10;272(2):1148–1155. doi: 10.1074/jbc.272.2.1148. [DOI] [PubMed] [Google Scholar]
  13. Klinedinst D. K., Drinkwater N. R. Mutagenesis by apurinic sites in normal and ataxia telangiectasia human lymphoblastoid cells. Mol Carcinog. 1992;6(1):32–42. doi: 10.1002/mc.2940060107. [DOI] [PubMed] [Google Scholar]
  14. Kubota Y., Nash R. A., Klungland A., Schär P., Barnes D. E., Lindahl T. Reconstitution of DNA base excision-repair with purified human proteins: interaction between DNA polymerase beta and the XRCC1 protein. EMBO J. 1996 Dec 2;15(23):6662–6670. [PMC free article] [PubMed] [Google Scholar]
  15. Lindahl T. Instability and decay of the primary structure of DNA. Nature. 1993 Apr 22;362(6422):709–715. doi: 10.1038/362709a0. [DOI] [PubMed] [Google Scholar]
  16. Lindahl T., Nyberg B. Heat-induced deamination of cytosine residues in deoxyribonucleic acid. Biochemistry. 1974 Jul 30;13(16):3405–3410. doi: 10.1021/bi00713a035. [DOI] [PubMed] [Google Scholar]
  17. Lindahl T. Repair of intrinsic DNA lesions. Mutat Res. 1990 May;238(3):305–311. doi: 10.1016/0165-1110(90)90022-4. [DOI] [PubMed] [Google Scholar]
  18. Matthews B. W. Solvent content of protein crystals. J Mol Biol. 1968 Apr 28;33(2):491–497. doi: 10.1016/0022-2836(68)90205-2. [DOI] [PubMed] [Google Scholar]
  19. McGeoch D. J., Dalrymple M. A., Davison A. J., Dolan A., Frame M. C., McNab D., Perry L. J., Scott J. E., Taylor P. The complete DNA sequence of the long unique region in the genome of herpes simplex virus type 1. J Gen Virol. 1988 Jul;69(Pt 7):1531–1574. doi: 10.1099/0022-1317-69-7-1531. [DOI] [PubMed] [Google Scholar]
  20. Mol C. D., Arvai A. S., Sanderson R. J., Slupphaug G., Kavli B., Krokan H. E., Mosbaugh D. W., Tainer J. A. Crystal structure of human uracil-DNA glycosylase in complex with a protein inhibitor: protein mimicry of DNA. Cell. 1995 Sep 8;82(5):701–708. doi: 10.1016/0092-8674(95)90467-0. [DOI] [PubMed] [Google Scholar]
  21. Mol C. D., Arvai A. S., Slupphaug G., Kavli B., Alseth I., Krokan H. E., Tainer J. A. Crystal structure and mutational analysis of human uracil-DNA glycosylase: structural basis for specificity and catalysis. Cell. 1995 Mar 24;80(6):869–878. doi: 10.1016/0092-8674(95)90290-2. [DOI] [PubMed] [Google Scholar]
  22. Nash H. M., Bruner S. D., Schärer O. D., Kawate T., Addona T. A., Spooner E., Lane W. S., Verdine G. L. Cloning of a yeast 8-oxoguanine DNA glycosylase reveals the existence of a base-excision DNA-repair protein superfamily. Curr Biol. 1996 Aug 1;6(8):968–980. doi: 10.1016/s0960-9822(02)00641-3. [DOI] [PubMed] [Google Scholar]
  23. Nicholl I. D., Nealon K., Kenny M. K. Reconstitution of human base excision repair with purified proteins. Biochemistry. 1997 Jun 17;36(24):7557–7566. doi: 10.1021/bi962950w. [DOI] [PubMed] [Google Scholar]
  24. Nilsen H., Yazdankhah S. P., Eftedal I., Krokan H. E. Sequence specificity for removal of uracil from U.A pairs and U.G mismatches by uracil-DNA glycosylase from Escherichia coli, and correlation with mutational hotspots. FEBS Lett. 1995 Apr 3;362(2):205–209. doi: 10.1016/0014-5793(95)00244-4. [DOI] [PubMed] [Google Scholar]
  25. Panayotou G., Brown T., Barlow T., Pearl L. H., Savva R. Direct measurement of the substrate preference of uracil-DNA glycosylase. J Biol Chem. 1998 Jan 2;273(1):45–50. doi: 10.1074/jbc.273.1.45. [DOI] [PubMed] [Google Scholar]
  26. Parikh S. S., Mol C. D., Tainer J. A. Base excision repair enzyme family portrait: integrating the structure and chemistry of an entire DNA repair pathway. Structure. 1997 Dec 15;5(12):1543–1550. doi: 10.1016/s0969-2126(97)00303-1. [DOI] [PubMed] [Google Scholar]
  27. Parkinson G., Vojtechovsky J., Clowney L., Brünger A. T., Berman H. M. New parameters for the refinement of nucleic acid-containing structures. Acta Crystallogr D Biol Crystallogr. 1996 Jan 1;52(Pt 1):57–64. doi: 10.1107/S0907444995011115. [DOI] [PubMed] [Google Scholar]
  28. Pourquier P., Ueng L. M., Kohlhagen G., Mazumder A., Gupta M., Kohn K. W., Pommier Y. Effects of uracil incorporation, DNA mismatches, and abasic sites on cleavage and religation activities of mammalian topoisomerase I. J Biol Chem. 1997 Mar 21;272(12):7792–7796. doi: 10.1074/jbc.272.12.7792. [DOI] [PubMed] [Google Scholar]
  29. Prasad R., Singhal R. K., Srivastava D. K., Molina J. T., Tomkinson A. E., Wilson S. H. Specific interaction of DNA polymerase beta and DNA ligase I in a multiprotein base excision repair complex from bovine testis. J Biol Chem. 1996 Jul 5;271(27):16000–16007. doi: 10.1074/jbc.271.27.16000. [DOI] [PubMed] [Google Scholar]
  30. Savva R., McAuley-Hecht K., Brown T., Pearl L. The structural basis of specific base-excision repair by uracil-DNA glycosylase. Nature. 1995 Feb 9;373(6514):487–493. doi: 10.1038/373487a0. [DOI] [PubMed] [Google Scholar]
  31. Slupphaug G., Eftedal I., Kavli B., Bharati S., Helle N. M., Haug T., Levine D. W., Krokan H. E. Properties of a recombinant human uracil-DNA glycosylase from the UNG gene and evidence that UNG encodes the major uracil-DNA glycosylase. Biochemistry. 1995 Jan 10;34(1):128–138. doi: 10.1021/bi00001a016. [DOI] [PubMed] [Google Scholar]
  32. Slupphaug G., Mol C. D., Kavli B., Arvai A. S., Krokan H. E., Tainer J. A. A nucleotide-flipping mechanism from the structure of human uracil-DNA glycosylase bound to DNA. Nature. 1996 Nov 7;384(6604):87–92. doi: 10.1038/384087a0. [DOI] [PubMed] [Google Scholar]
  33. Svendsen P. C., Yee H. A., Winkfein R. J., van de Sande J. H. The mouse uracil-DNA glycosylase gene: isolation of cDNA and genomic clones and mapping ung to mouse chromosome 5. Gene. 1997 Apr 21;189(2):175–181. doi: 10.1016/s0378-1119(96)00797-4. [DOI] [PubMed] [Google Scholar]
  34. Thayer M. M., Ahern H., Xing D., Cunningham R. P., Tainer J. A. Novel DNA binding motifs in the DNA repair enzyme endonuclease III crystal structure. EMBO J. 1995 Aug 15;14(16):4108–4120. doi: 10.1002/j.1460-2075.1995.tb00083.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Varshney U., Hutcheon T., van de Sande J. H. Sequence analysis, expression, and conservation of Escherichia coli uracil DNA glycosylase and its gene (ung). J Biol Chem. 1988 Jun 5;263(16):7776–7784. [PubMed] [Google Scholar]
  36. Varshney U., van de Sande J. H. Specificities and kinetics of uracil excision from uracil-containing DNA oligomers by Escherichia coli uracil DNA glycosylase. Biochemistry. 1991 Apr 23;30(16):4055–4061. doi: 10.1021/bi00230a033. [DOI] [PubMed] [Google Scholar]
  37. Vassylyev D. G., Morikawa K. Precluding uracil from DNA. Structure. 1996 Dec 15;4(12):1381–1385. doi: 10.1016/s0969-2126(96)00145-1. [DOI] [PubMed] [Google Scholar]
  38. Verdine G. L., Bruner S. D. How do DNA repair proteins locate damaged bases in the genome? Chem Biol. 1997 May;4(5):329–334. doi: 10.1016/s1074-5521(97)90123-x. [DOI] [PubMed] [Google Scholar]
  39. Wilson D. M., 3rd, Takeshita M., Demple B. Abasic site binding by the human apurinic endonuclease, Ape, and determination of the DNA contact sites. Nucleic Acids Res. 1997 Mar 1;25(5):933–939. doi: 10.1093/nar/25.5.933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Xiao W., Samson L. In vivo evidence for endogenous DNA alkylation damage as a source of spontaneous mutation in eukaryotic cells. Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2117–2121. doi: 10.1073/pnas.90.6.2117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Zhou W., Doetsch P. W. Effects of abasic sites and DNA single-strand breaks on prokaryotic RNA polymerases. Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6601–6605. doi: 10.1073/pnas.90.14.6601. [DOI] [PMC free article] [PubMed] [Google Scholar]

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