Recognition of DNA Adducts by Human Nucleotide Excision Repair
1996; Elsevier BV; Volume: 271; Issue: 41 Linguagem: Inglês
10.1074/jbc.271.41.25089
ISSN1083-351X
AutoresDaniela Gunz, Martin Heß, Hanspeter Naegeli,
Tópico(s)Advanced biosensing and bioanalysis techniques
ResumoThe mechanism by which mammalian nucleotide excision repair (NER) detects a wide range of base lesions is poorly understood. Here, we tested the ability of human NER to recognize bulky modifications that either destabilize the DNA double helix (acetylaminofluorene (AAF) and benzo[a]pyrene diol-epoxide (BPDE) adducts, UV radiation products) or induce opposite effects by stabilizing the double helix (8-methoxypsoralen (8-MOP), anthramycin, and CC-1065 adducts). We constructed plasmid DNA carrying a defined number of each of these adducts and determined their potential to sequester NER factors contained in a human cell-free extract. For that purpose, we measured the capacity of damaged plasmids to compete with excision repair of a site-directed NER substrate. This novel approach showed differences of more than 3 orders of magnitude in the efficiency by which helix-destabilizing and helix-stabilizing adducts sequester NER factors. For example, AAF modifications were able to compete with the NER substrate ~1740 times more effectively than 8-MOP adducts. The sequestration potency decreased with the following order of adducts, AAF > UV ≥ BPDE > 8-MOP > anthramycin, CC-1065. A strong preference for helix-destabilizing lesions was confirmed by monitoring the formation of NER patches at site-specific adducts with either AAF or CC-1065. This comparison based on factor sequestration and repair synthesis indicates that human NER is primarily targeted to sites at which the secondary structure of DNA is destabilized. Thus, an early step of DNA damage recognition involves thermodynamic probing of the duplex. The mechanism by which mammalian nucleotide excision repair (NER) detects a wide range of base lesions is poorly understood. Here, we tested the ability of human NER to recognize bulky modifications that either destabilize the DNA double helix (acetylaminofluorene (AAF) and benzo[a]pyrene diol-epoxide (BPDE) adducts, UV radiation products) or induce opposite effects by stabilizing the double helix (8-methoxypsoralen (8-MOP), anthramycin, and CC-1065 adducts). We constructed plasmid DNA carrying a defined number of each of these adducts and determined their potential to sequester NER factors contained in a human cell-free extract. For that purpose, we measured the capacity of damaged plasmids to compete with excision repair of a site-directed NER substrate. This novel approach showed differences of more than 3 orders of magnitude in the efficiency by which helix-destabilizing and helix-stabilizing adducts sequester NER factors. For example, AAF modifications were able to compete with the NER substrate ~1740 times more effectively than 8-MOP adducts. The sequestration potency decreased with the following order of adducts, AAF > UV ≥ BPDE > 8-MOP > anthramycin, CC-1065. A strong preference for helix-destabilizing lesions was confirmed by monitoring the formation of NER patches at site-specific adducts with either AAF or CC-1065. This comparison based on factor sequestration and repair synthesis indicates that human NER is primarily targeted to sites at which the secondary structure of DNA is destabilized. Thus, an early step of DNA damage recognition involves thermodynamic probing of the duplex. INTRODUCTIONNucleotide excision repair (NER) 1The abbreviations used are: NERnucleotide excision repairAAFN-acetyl-2-aminofluoreneBPDEbenzo[a]pyrene diol-epoxide8-MOP8-methoxypsoralenRPAreplication protein AUVultravioletXPxeroderma pigmentosumpyrimidine(6-4) photoproduct6-(1,2)-dihydro-2-oxo-4-pyrimidyl)-5-methyl-2,4-(1H,3H) photoproduct. is an essential pathway for removing bulky base modifications from DNA. This repair mechanism involves endonucleolytic cleavage at two phosphodiester bonds, one 3ʹ and the other 5ʹ of the site of damage, followed by excision of DNA damage as the component of a single-stranded fragment (1Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. American Society for Microbiology, Washington, D. C.1995: 283Google Scholar, 2Sancar A. J. Biol. Chem. 1995; 270: 15915-15918Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 3Grossman L. Thiangalingam S. J. Biol. Chem. 1993; 268: 16871-16874Abstract Full Text PDF PubMed Google Scholar, 4Tanaka K. Wood R.D. Trends Biochem. Sci. 1994; 19: 83-86Abstract Full Text PDF PubMed Scopus (87) Google Scholar, 5Van Houten B. Microbiol. Rev. 1990; 54: 18-51Crossref PubMed Google Scholar). The excised oligonucleotide is replaced by DNA repair synthesis, and DNA continuity is reestablished by ligation. In mammalian cells, the major sites of incision are at the 5th phosphodiester bond 3ʹ and the 24th phosphodiester bond 5ʹ to the lesion (6Huang J.-C. Svoboda D.L. Reardon J.T. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3664-3668Crossref PubMed Scopus (375) Google Scholar).Human patients deficient in NER suffer from xeroderma pigmentosum (XP), a hereditary disease characterized by photosensitivity, increased incidence of skin cancer, and frequently neurological abnormalities (7Cleaver J.E. Kraemer K.H. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Disease. McGraw-Hill Inc., New York1989: 2949Google Scholar, 8Hoeijmakers J.H.J. Eur. J. Cancer. 1994; 30: 1912-1921Abstract Full Text PDF Scopus (112) Google Scholar). At the biochemical level, XP individuals are impaired in the removal of radiation products induced by the UV component of sunlight (9Cleaver J.E. Nature. 1968; 218: 652-656Crossref PubMed Scopus (1265) Google Scholar). Somatic cells obtained from these patients are also defective in excision repair of bulky DNA adducts resulting from genotoxic chemicals (1Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. American Society for Microbiology, Washington, D. C.1995: 283Google Scholar, 2Sancar A. J. Biol. Chem. 1995; 270: 15915-15918Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 4Tanaka K. Wood R.D. Trends Biochem. Sci. 1994; 19: 83-86Abstract Full Text PDF PubMed Scopus (87) Google Scholar). Recent in vitro studies using cell-free extracts showed that the range of base lesions processed by mammalian NER extends to nonbulky adducts, and even abasic sites are susceptible to excision repair by this pathway (10Huang J.-C. Hsu D.S. Kazantsev A. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12213-12217Crossref PubMed Scopus (211) Google Scholar).Biochemical reconstitution experiments demonstrated that mammalian NER is catalyzed by the coordinated action of at least 30 polypeptides (11Aboussekhra A. Biggerstaff M. Shivji M.K.K. Vilpo J.A. Moncollin V. Podust V.A. Protic Hübscher U. Egly J.-M. Wood R.D. Cell. 1995; 80: 859-868Abstract Full Text PDF PubMed Scopus (748) Google Scholar, 12Mu D. Park C.-H. Matsunaga T. Hsu D.S. Reardon J.T. Sancar A. J. Biol. Chem. 1995; 270: 2415-2418Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar). Several of these factors have been implicated in the recognition step of NER, primarily a complex made up of XPA and the three subunits of RPA (p70, p34, p11) (13He Z. Henricksen L.A. Wold M.S. Ingles C.J. Nature. 1995; 374: 566-569Crossref PubMed Scopus (371) Google Scholar); in addition, the multisubunit protein complex TFIIH and XPE protein may participate in damage recognition (1Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. American Society for Microbiology, Washington, D. C.1995: 283Google Scholar, 2Sancar A. J. Biol. Chem. 1995; 270: 15915-15918Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 3Grossman L. Thiangalingam S. J. Biol. Chem. 1993; 268: 16871-16874Abstract Full Text PDF PubMed Google Scholar, 4Tanaka K. Wood R.D. Trends Biochem. Sci. 1994; 19: 83-86Abstract Full Text PDF PubMed Scopus (87) Google Scholar). The molecular mechanism by which these proteins discriminate a large number of chemically unrelated DNA lesions as substrates for NER is unknown. However, the versatility of NER led to the assumption that this system recognizes conformational changes imposed on DNA at sites of damage rather than specific base modifications (2Sancar A. J. Biol. Chem. 1995; 270: 15915-15918Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 5Van Houten B. Microbiol. Rev. 1990; 54: 18-51Crossref PubMed Google Scholar). In this report, we compared recognition of DNA adducts where detailed structural information is available and identified a molecular determinant triggering initiation of the mammalian NER pathway.The acetylaminofluorene (AAF), benzo[a]pyrene diol-epoxide (BPDE), 8-methoxypsoralen (8-MOP), anthramycin, and CC-1065 moieties are illustrated in Fig. 1. Melting temperature studies have shown that these bulky base adducts alter the thermodynamic characteristics of DNA in different ways. AAF adducts (14O'Handley S.F. Sanford S.G. Xu R. Lester C.C. Hingerty B.E. Broyde S. Krugh T.R. Biochemistry. 1993; 32: 2481-2497Crossref PubMed Scopus (141) Google Scholar, 15Garcia A. Lambert I.B. Fuchs R.P.P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5989-5993Crossref PubMed Scopus (80) Google Scholar), BPDE adducts (16Zou Y. Liu T.-M. Geacintov N.E. Van Houten B. Biochemistry. 1995; 34: 13582-13593Crossref PubMed Scopus (77) Google Scholar, 17Xu R. Birke S. Carberry S.E. Geacintov N.E. Swenberg C.E. Harvey R.G. Nucleic Acids Res. 1992; 20: 6167-6176Crossref PubMed Scopus (22) Google Scholar) and UV radiation products (18Rahn R.O. Patrick M.H. Wang S.Y. Photochemistry and Photobiology of Nucleic Acids. Vol 2. Academic Press, NY1976: 97Google Scholar) destabilize the DNA double helix relative to nonmodified DNA, whereas 8-MOP (19Shi Y.-B. Hearst J.E. Biochemistry. 1986; 25: 5895-5902Crossref PubMed Scopus (55) Google Scholar, 20Shi Y.-B Griffith J. Hearst J.E. Nucleic Acids Res. 1988; 16: 8945-8952Crossref PubMed Scopus (16) Google Scholar), anthramycin (21Hurley L.H. Thurston D.E. Pharmacol. Res. 1984; 1: 53-59Crossref Scopus (61) Google Scholar), and CC-1065 adducts (22Reynolds V.L. McGovren J.P. Hurley L.H. J. Antibiot. (Tokyo). 1986; 39: 319-334Crossref PubMed Scopus (154) Google Scholar) stabilize the DNA duplex. In the predominant adduct formed by N-acetoxy-2-acetylaminofluorene (AAF-C8-guanine), the modified base is rotated out of the helix axis, and the duplex is locally denatured (14O'Handley S.F. Sanford S.G. Xu R. Lester C.C. Hingerty B.E. Broyde S. Krugh T.R. Biochemistry. 1993; 32: 2481-2497Crossref PubMed Scopus (141) Google Scholar, 23Schwartz A. Marrot L. Leng M. J. Mol. Biol. 1989; 207: 445-450Crossref PubMed Scopus (22) Google Scholar). Reaction of anti-7,8-diol 9,10-epoxy-benzo[a]pyrene with double-stranded DNA generates mainly (+)-trans-anti-BPDE-N2-guanine adducts with quantitatively minor lesions resulting from (-)-trans, (+)-cis, and (-)-cis additions to the same position N2 of guanine (24de los Santos C. Cosman M. Hingerty B.E. Ibanez V. Margulis L.A. Geacintov N.E. Broyde S. Patel D.J. Biochemistry. 1992; 31: 5245-5252Crossref PubMed Scopus (185) Google Scholar, 25Cosman M. de los Santos C. Fiala R. Hingerty B.E. Ibanez V. Luna E. Harvey R. Geacintov N.E. Broyde S. Patel D.J. Biochemistry. 1993; 32: 4145-4155Crossref PubMed Scopus (164) Google Scholar). Depending on their stereochemistry, these BPDE-N2-guanine adducts are either accommodated in the minor groove (24de los Santos C. Cosman M. Hingerty B.E. Ibanez V. Margulis L.A. Geacintov N.E. Broyde S. Patel D.J. Biochemistry. 1992; 31: 5245-5252Crossref PubMed Scopus (185) Google Scholar) and cause DNA unwinding (17Xu R. Birke S. Carberry S.E. Geacintov N.E. Swenberg C.E. Harvey R.G. Nucleic Acids Res. 1992; 20: 6167-6176Crossref PubMed Scopus (22) Google Scholar) or assume a base-displacement configuration with localized base pair disruption (25Cosman M. de los Santos C. Fiala R. Hingerty B.E. Ibanez V. Luna E. Harvey R. Geacintov N.E. Broyde S. Patel D.J. Biochemistry. 1993; 32: 4145-4155Crossref PubMed Scopus (164) Google Scholar). For comparison, we also generated helix-destabilizing modifications by irradiating DNA with UV light at 254 nm, producing as major lesions cyclobutane pyrimidine dimers and pyrimidine(6-4) photoproducts in a ratio of about 3:1 (26Cadet J. Anselmino C. Douki T. Voituriez L. J. Photochem. Photobiol. B Biol. 1992; 15: 277-298Crossref PubMed Scopus (184) Google Scholar).Treatment of DNA with 8-MOP and long wavelength UV light (>320 nm) yields psoralen monoadducts and a small proportion of psoralen diadducts (27Hearst J.E. Isaacs S.T. Kanne D. Rapoport H. Straub K. Q. Rev. Biophys. 1984; 17: 1-44Crossref PubMed Scopus (137) Google Scholar). This photoaddition reaction occurs between the 5,6 double bond of pyrimidine bases and either the 3,4 (pyrone) or the 4ʹ,5ʹ (furan) double bond of the psoralen. Modifications with psoralen induce helical distortion by unwinding the duplex and enhancing backbone flexibility (28Spielmann H.P. Dwyer T.J. Hearst J.E. Wemmer D.E. Biochemistry. 1995; 34: 12937-12953Crossref PubMed Scopus (70) Google Scholar) but fail to destabilize the secondary structure of DNA. On the contrary, thermostability measurements showed that both pyroneside and furanside monoadducts stabilize the helix by mediating stacking interactions between the psoralen moiety and the surrounding base pairs (19Shi Y.-B. Hearst J.E. Biochemistry. 1986; 25: 5895-5902Crossref PubMed Scopus (55) Google Scholar, 20Shi Y.-B Griffith J. Hearst J.E. Nucleic Acids Res. 1988; 16: 8945-8952Crossref PubMed Scopus (16) Google Scholar, 28Spielmann H.P. Dwyer T.J. Hearst J.E. Wemmer D.E. Biochemistry. 1995; 34: 12937-12953Crossref PubMed Scopus (70) Google Scholar). Helix-stabilizing adducts were also obtained using anthramycin, a pyrrolo[1,4]benzodiazepine antibiotic, and CC-1065, a composite compound consisting of three pyrroloindole subunits joined by amide linkages (Fig. 1). Anthramycin binds selectively to N2 of guanine through aminal bonds and forms covalent adducts with essentially no distortion of the DNA helix (21Hurley L.H. Thurston D.E. Pharmacol. Res. 1984; 1: 53-59Crossref Scopus (61) Google Scholar, 29Krugh T.R. Graves D.E. Stone M. Biochemistry. 1989; 28: 9988-9994Crossref PubMed Scopus (32) Google Scholar). CC-1065 displays a cyclopropyl ring that alkylates DNA at position N3 of adenine, generating covalent adducts that cause bending and winding of the double helix (22Reynolds V.L. McGovren J.P. Hurley L.H. J. Antibiot. (Tokyo). 1986; 39: 319-334Crossref PubMed Scopus (154) Google Scholar, 30Lee C.-S. Sun D. Kizu R. Hurley L.H. Chem. Res. Toxicol. 1991; 4: 203-213Crossref PubMed Scopus (56) Google Scholar). Both anthramycin and CC-1065 adducts enhance duplex stability through noncovalent interactions derived from hydrogen bonds (anthramycin) or van der Waals and hydrophobic forces (CC-1065) within the minor groove of DNA (21Hurley L.H. Thurston D.E. Pharmacol. Res. 1984; 1: 53-59Crossref Scopus (61) Google Scholar, 22Reynolds V.L. McGovren J.P. Hurley L.H. J. Antibiot. (Tokyo). 1986; 39: 319-334Crossref PubMed Scopus (154) Google Scholar, 30Lee C.-S. Sun D. Kizu R. Hurley L.H. Chem. Res. Toxicol. 1991; 4: 203-213Crossref PubMed Scopus (56) Google Scholar).Recognition of these bulky adducts was compared by monitoring their capacity to sequester NER factors. To that end, we have developed a competition assay that measures the efficiency by which damaged plasmids compete for NER factors operating on a site-directed substrate (31Hess M.T. Gunz D. Naegeli H. Nucleic Acids Res. 1996; 24: 824-828Crossref PubMed Scopus (21) Google Scholar). As a source of NER activity we exploited a standard soluble extract from human cells (32Manley J.L. Fire A. Samuels M. Sharp P.A. Methods Enzymol. 1983; 101: 568-582Crossref PubMed Scopus (222) Google Scholar, 33Wood R.D. Robins P. Lindahl T. Cell. 1988; 53: 97-106Abstract Full Text PDF PubMed Scopus (380) Google Scholar). Under the conditions used in our study, this cell-free extract does not support chromatin assembly or transcription, thereby eliminating nuclear activities that modulate the intrinsic capacity of NER to recognize DNA damage (34Wood R.D. Coverley D. BioEssays. 1991; 13: 447-453Crossref PubMed Scopus (27) Google Scholar, 35Hanawalt P.C. Science. 1994; 266: 1957-1958Crossref PubMed Scopus (452) Google Scholar). This system revealed >1000-fold differences in the capacity of the tested adducts to sequester human NER factors. Those lesions that destabilize the DNA helix (AAF or BPDE adducts, UV radiation products) were effective competitors. In contrast, those adducts that stabilize the helix (8-MOP, anthramycin, and CC-1065 adducts) displayed minimal competing effects. In parallel, site-specifically placed CC-1065 adducts were unable to detectably stimulate synthesis of DNA repair patches. These results indicate that an early subset of NER recognition factors is attracted to structural defects associated with unfavorable thermodynamic changes of the DNA double helix. As a consequence, human NER is preferentially targeted to sites of helical instability.DISCUSSIONMammalian NER processes a wide range of chemically and structurally distinct base adducts, but some types of damage are repaired at higher rates than others (1Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. American Society for Microbiology, Washington, D. C.1995: 283Google Scholar, 2Sancar A. J. Biol. Chem. 1995; 270: 15915-15918Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 10Huang J.-C. Hsu D.S. Kazantsev A. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12213-12217Crossref PubMed Scopus (211) Google Scholar, 45Mitchell D.L. Photochem. Photobiol. 1988; 48: 51-57Crossref PubMed Scopus (308) Google Scholar, 46Szymkowski D.E. Lawrence C.W. Wood R.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9823-9827Crossref PubMed Scopus (79) Google Scholar). The biochemical mechanisms underlying this broad and extremely heterogeneous response are poorly understood. In the present study, we have established a structure-activity relationship with respect to DNA damage recognition using a novel in vitro approach based on NER factor sequestration. Our assay requires highly purified plasmid DNA (pUC19) containing a defined number of a particular base adduct and measures the efficiency by which these damaged DNA molecules sequester human NER factors and, hence, inhibit repair of a site-directed NER substrate (Fig. 9). Using this competition assay, we have tested recognition of several bulky base adducts that are known to display potent mutagenic and/or carcinogenic effects and of which detailed structural information is available. The susceptibility of some of these bulky adducts (BPDE, anthramycin, CC-1065) to mammalian NER has not been examined in a defined in vitro system before.Fig. 9Basic design of the repair competition assay. This assay was used to assess the capacity of damaged pUC19 to sequester NER factors contained in the HeLa cell extract. Sequestration was measured by monitoring the competitive inhibition of NER operating on a site-directed AAF-guanine adduct in M13 DNA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Several observations led us to exploit the repair competition assay to compare damage recognition by the human NER system. First, excision repair of the site-directed AAF adduct used as a substrate in these assays is strictly dependent on the NER pathway. In fact, essentially no synthesis of DNA repair patches was observed when the site-directed substrate was incubated with extracts from NER-deficient XP-A or XP-C cell lines, but DNA repair synthesis was reconstituted by combining the two different extracts (Fig. 3B). Second, competitor pUC19 containing AAF adducts (Fig. 4), UV radiation products (31Hess M.T. Gunz D. Naegeli H. Nucleic Acids Res. 1996; 24: 824-828Crossref PubMed Scopus (21) Google Scholar), BPDE adducts (Fig. 5), or 8-MOP adducts (Fig. 6) inhibited NER of the site-specific substrate in a dose-dependent manner. When AAF-damaged competitor was tested, we found that NER of the site-specific AAF adduct on the substrate was inhibited to 50% at a 1:1 stoichiometry of these lesions (Fig. 4), indicating that competition for NER factors occurs in a quantitative manner. Third, nondamaged pUC19 DNA caused only marginal inhibition of NER operating on the substrate. Conversely, efficient competitors such as AAF-damaged pUC19 suppressed NER activity in the covalently modified region of the substrate, without significantly reducing nonspecific nucleotide incorporation in an adjacent DNA segment (Fig. 4). Thus, the competition effect is selective for NER activity. Fourth, the principal finding obtained in the competition assay (differential recognition of thermodynamically diverse lesions) was confirmed by comparing DNA repair synthesis induced by site-specific adducts with either a helix-destabilizing (AAF) or helix-stabilizing (CC-1065) compound (Fig. 8). Finally, previous studies have shown that damage recognition/DNA incision constitute the rate-limiting step of NER in the cell-free extract (47Hansson J. Munn M. Rupp W.D. Kahn R. Wood R.D. J. Biol. Chem. 1989; 264: 21788-21792Abstract Full Text PDF PubMed Google Scholar), indicating that competition for repair factors is likely to occur at an early, preincisional level of the pathway.The repair competition data demonstrated that DNA conformation at the site of damage is of critical importance for recognition. The capacity of the tested adducts to sequester human NER factors decreased with the following order: AAF > UV radiation products ≥ BPDE > 8-MOP > anthramycin, CC-1065. The competition exerted by AAF, UV, and BPDE adducts was 2-3 orders of magnitude stronger than the competition effected by 8-MOP, anthramycin, or CC-1065 adducts. For example, AAF modifications were able to sequester NER factors 1740 times more efficiently than 8-MOP adducts. This striking hierarchy of sequestration efficiency, combined with the known thermodynamic characteristics of the tested lesions, indicates that mammalian NER is primarily targeted to structural defects that destabilize the double-helical conformation of DNA. In fact, AAF modifications have been shown to completely abolish base pairing between the adducted guanine and its complementary cytosine (14O'Handley S.F. Sanford S.G. Xu R. Lester C.C. Hingerty B.E. Broyde S. Krugh T.R. Biochemistry. 1993; 32: 2481-2497Crossref PubMed Scopus (141) Google Scholar, 15Garcia A. Lambert I.B. Fuchs R.P.P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5989-5993Crossref PubMed Scopus (80) Google Scholar). Analysis with chloroacetaldehyde or osmium tetroxide revealed that this helix destabilizing effect is not limited to the site of AAF modification but rather extends to the neighboring base pairs in the duplex (23Schwartz A. Marrot L. Leng M. J. Mol. Biol. 1989; 207: 445-450Crossref PubMed Scopus (22) Google Scholar). In comparison, BPDE-N2-guanine adducts exert more moderate effects on double-helical stability (16Zou Y. Liu T.-M. Geacintov N.E. Van Houten B. Biochemistry. 1995; 34: 13582-13593Crossref PubMed Scopus (77) Google Scholar, 17Xu R. Birke S. Carberry S.E. Geacintov N.E. Swenberg C.E. Harvey R.G. Nucleic Acids Res. 1992; 20: 6167-6176Crossref PubMed Scopus (22) Google Scholar, 24de los Santos C. Cosman M. Hingerty B.E. Ibanez V. Margulis L.A. Geacintov N.E. Broyde S. Patel D.J. Biochemistry. 1992; 31: 5245-5252Crossref PubMed Scopus (185) Google Scholar). Psoralen monoadducts, on the other hand, slightly increase helical stability (19Shi Y.-B. Hearst J.E. Biochemistry. 1986; 25: 5895-5902Crossref PubMed Scopus (55) Google Scholar, 20Shi Y.-B Griffith J. Hearst J.E. Nucleic Acids Res. 1988; 16: 8945-8952Crossref PubMed Scopus (16) Google Scholar, 28Spielmann H.P. Dwyer T.J. Hearst J.E. Wemmer D.E. Biochemistry. 1995; 34: 12937-12953Crossref PubMed Scopus (70) Google Scholar), whereas anthramycin and CC-1065 adducts produce substantial increments in helical stability (21Hurley L.H. Thurston D.E. Pharmacol. Res. 1984; 1: 53-59Crossref Scopus (61) Google Scholar, 22Reynolds V.L. McGovren J.P. Hurley L.H. J. Antibiot. (Tokyo). 1986; 39: 319-334Crossref PubMed Scopus (154) Google Scholar). Thus, an early subset of NER factors appears to be endowed with the capacity to sense thermodynamic parameters of double-stranded DNA and preferentially interact with those sites that exhibit unfavorable changes in free energy. Among the known NER factors, the XPA-RPA complex is a possible protein candidate for executing this recognition function involving thermodynamic probing of DNA. This hypothesis is prompted by previous reports demonstrating that XPA stimulates RPA's single-stranded DNA binding activity (48Lee S.-H. Kim D.-K. Drissi R. J. Biol. Chem. 1995; 270: 21800-21805Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) and that the two factors display strong cooperativity in binding to AAF- or UV-damaged DNA (13He Z. Henricksen L.A. Wold M.S. Ingles C.J. Nature. 1995; 374: 566-569Crossref PubMed Scopus (371) Google Scholar, 49Li L. Lu X. Peterson C.A. Legerski R.J. Mol. Cell. Biol. 1995; 15: 5396-5402Crossref PubMed Scopus (225) Google Scholar).Several previous reports related to damage recognition in mammalian NER support the concept of thermodynamic recognition. For example, cyclobutane pyrimidine dimers are processed in vivo and in vitro at a considerably lower rate than pyrimidine(6-4) photoproducts (45Mitchell D.L. Photochem. Photobiol. 1988; 48: 51-57Crossref PubMed Scopus (308) Google Scholar, 46Szymkowski D.E. Lawrence C.W. Wood R.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9823-9827Crossref PubMed Scopus (79) Google Scholar, 50Suquet C. Mitchell D.L. Smerdon M.J. J. Biol. Chem. 1995; 270: 16507-16509Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The thermodynamic properties of DNA fragments containing a cyclobutane dimer indicate that this lesion minimally affects the ability to form a duplex (51Taylor J.-S. Garrett D.S. Brockie I.R. Svoboda D.L. Telser J. Biochemistry. 1990; 29: 8858-8866Crossref PubMed Scopus (110) Google Scholar), whereas pyrimidine(6-4) photoproducts strongly favor disruption of the double helix (52Kim J.-K. Choi B.-S. Eur. J. Biochem. 1995; 228: 849-854Crossref PubMed Scopus (102) Google Scholar). The different stereoisomers of BPDE-N2-guanine have distinct effects on the stability of a short DNA duplex, the (+)-trans adduct being more helix-destabilizing than the (+)-cis isomer (16Zou Y. Liu T.-M. Geacintov N.E. Van Houten B. Biochemistry. 1995; 34: 13582-13593Crossref PubMed Scopus (77) Google Scholar). Consistent with the concept of thermodynamic recognition, a recent analysis of epidermal tissue obtained from benzo[a]pyrene-treated mice showed that (+)-trans-anti-BPDE-N2-guanine adducts are repaired about three times faster than (+)-cis-anti-BPDE-N2-guanine adducts in vivo (53Suh M. Ariese F. Small G.J. Jankowiak R. Hewer A. Phillips D.H. Carcinogenesis. 1995; 16: 2561-2569Crossref PubMed Scopus (55) Google Scholar). Thermodynamic recognition is also consistent with the finding that certain nonbulky adducts, primarily abasic sites, constitute efficient substrates for human NER (10Huang J.-C. Hsu D.S. Kazantsev A. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12213-12217Crossref PubMed Scopus (211) Google Scholar). In fact, the loss of a base from DNA inherently destabilizes the duplex (23Schwartz A. Marrot L. Leng M. J. Mol. Biol. 1989; 207: 445-450Crossref PubMed Scopus (22) Google Scholar, 54Kalnik M.W. Chang C.-N. Johnson F. Grollman A.P. Patel D.J. Biochemistry. 1989; 28: 3373-3383Crossref PubMed Scopus (69) Google Scholar).Interestingly, the thermodynamic characteristics of damaged DNA are less important for recognition by (A)BC excinuclease, the prokaryotic NER system. For example, it was found that (A)BC excinuclease incises at (+)-cis-anti-BPDE-N2-guanine adducts more efficiently than at (+)-trans-anti-BPDE-N2-guanine adducts (16Zou Y. Liu T.-M. Geacintov N.E. Van Houten B. Biochemistry. 1995; 34: 13582-13593Crossref PubMed Scopus (77) Google Scholar), which is exactly the opposite of what was observed in mammalian tissues (53Suh M. Ariese F. Small G.J. Jankowiak R. Hewer A. Phillips D.H. Carcinogenesis. 1995; 16: 2561-2569Crossref PubMed Scopus (55) Google Scholar). Similarly, the helix-stabilizing psoralen monoadduct is an efficient substrate for the (A)BC excinuclease (10Huang J.-C. Hsu D.S. Kazantsev A. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12213-12217Crossref PubMed Scopus (211) Google Scholar) but constitutes a modest substrate for the human NER system (10Huang J.-C. Hsu D.S. Kazantsev A. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12213-12217Crossref PubMed Scopus (211) Google Scholar and this study). Also, the poor sequestration capacity of anthramycin-DNA adducts observed in our competition assay using the mammalian system contrasts with a previous report indicating that prokaryotic (A)BC excinuclease processes anthramycin-containing DNA with a four to five times higher rate than UV-irradiated DNA (55Nazimiec M. Grossman L. Tang M. J. Biol. Chem. 1992; 267: 24716-24724Abstract Full Text PDF PubMed Google Scholar). A diverging substrate preference between prokaryotic and mammalian NER has already been noted before (10Huang J.-C. Hsu D.S. Kazantsev A. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12213-12217Crossref PubMed Scopus (211) Google Scholar) and appears to reflect the lack of homology between the two systems.Damage recognition in NER has been proposed to occur in a sequence of partially overlapping biochemical reactions, each of which confers increased selectivity for damaged sites (56Lin J.-J. Sancar A. Mol. Microbiol. 1992; 6: 2219-2224Crossref PubMed Scopus (91) Google Scholar). The present report shows that human NER recognition factors are preferentially targeted to those types of DNA damage that destabilize the duplex. This observation indicates that thermodynamic probing of DNA stability constitutes an early step in damage recognition by mammalian NER. A thermodynamically biased recognition mechanism may serve, in the context of the large mammalian genome, to locate with highest priority those lesions with more pronounced cytotoxic and mutagenic potentials. INTRODUCTIONNucleotide excision repair (NER) 1The abbreviations used are: NERnucleotide excision repairAAFN-acetyl-2-aminofluoreneBPDEbenzo[a]pyrene diol-epoxide8-MOP8-methoxypsoralenRPAreplication protein AUVultravioletXPxeroderma pigmentosumpyrimidine(6-4) photoproduct6-(1,2)-dihydro-2-oxo-4-pyrimidyl)-5-methyl-2,4-(1H,3H) photoproduct. is an essential pathway for removing bulky base modifications from DNA. This repair mechanism involves endonucleolytic cleavage at two phosphodiester bonds, one 3ʹ and the other 5ʹ of the site of damage, followed by excision of DNA damage as the component of a single-stranded fragment (1Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. American Society for Microbiology, Washington, D. C.1995: 283Google Scholar, 2Sancar A. J. Biol. Chem. 1995; 270: 15915-15918Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 3Grossman L. Thiangalingam S. J. Biol. Chem. 1993; 268: 16871-16874Abstract Full Text PDF PubMed Google Scholar, 4Tanaka K. Wood R.D. Trends Biochem. Sci. 1994; 19: 83-86Abstract Full Text PDF PubMed Scopus (87) Google Scholar, 5Van Houten B. Microbiol. Rev. 1990; 54: 18-51Crossref PubMed Google Scholar). The excised oligonucleotide is replaced by DNA repair synthesis, and DNA continuity is reestablished by ligation. In mammalian cells, the major sites of incision are at the 5th phosphodiester bond 3ʹ and the 24th phosphodiester bond 5ʹ to the lesion (6Huang J.-C. Svoboda D.L. Reardon J.T. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3664-3668Crossref PubMed Scopus (375) Google Scholar).Human patients deficient in NER suffer from xeroderma pigmentosum (XP), a hereditary disease characterized by photosensitivity, increased incidence of skin cancer, and frequently neurological abnormalities (7Cleaver J.E. Kraemer K.H. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Disease. McGraw-Hill Inc., New York1989: 2949Google Scholar, 8Hoeijmakers J.H.J. Eur. J. Cancer. 1994; 30: 1912-1921Abstract Full Text PDF Scopus (112) Google Scholar). At the biochemical level, XP individuals are impaired in the removal of radiation products induced by the UV component of sunlight (9Cleaver J.E. Nature. 1968; 218: 652-656Crossref PubMed Scopus (1265) Google Scholar). Somatic cells obtained from these patients are also defective in excision repair of bulky DNA adducts resulting from genotoxic chemicals (1Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. American Society for Microbiology, Washington, D. C.1995: 283Google Scholar, 2Sancar A. J. Biol. Chem. 1995; 270: 15915-15918Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 4Tanaka K. Wood R.D. Trends Biochem. Sci. 1994; 19: 83-86Abstract Full Text PDF PubMed Scopus (87) Google Scholar). Recent in vitro studies using cell-free extracts showed that the range of base lesions processed by mammalian NER extends to nonbulky adducts, and even abasic sites are susceptible to excision repair by this pathway (10Huang J.-C. Hsu D.S. Kazantsev A. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12213-12217Crossref PubMed Scopus (211) Google Scholar).Biochemical reconstitution experiments demonstrated that mammalian NER is catalyzed by the coordinated action of at least 30 polypeptides (11Aboussekhra A. Biggerstaff M. Shivji M.K.K. Vilpo J.A. Moncollin V. Podust V.A. Protic Hübscher U. Egly J.-M. Wood R.D. Cell. 1995; 80: 859-868Abstract Full Text PDF PubMed Scopus (748) Google Scholar, 12Mu D. Park C.-H. Matsunaga T. Hsu D.S. Reardon J.T. Sancar A. J. Biol. Chem. 1995; 270: 2415-2418Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar). Several of these factors have been implicated in the recognition step of NER, primarily a complex made up of XPA and the three subunits of RPA (p70, p34, p11) (13He Z. Henricksen L.A. Wold M.S. Ingles C.J. Nature. 1995; 374: 566-569Crossref PubMed Scopus (371) Google Scholar); in addition, the multisubunit protein complex TFIIH and XPE protein may participate in damage recognition (1Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. American Society for Microbiology, Washington, D. C.1995: 283Google Scholar, 2Sancar A. J. Biol. Chem. 1995; 270: 15915-15918Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 3Grossman L. Thiangalingam S. J. Biol. Chem. 1993; 268: 16871-16874Abstract Full Text PDF PubMed Google Scholar, 4Tanaka K. Wood R.D. Trends Biochem. Sci. 1994; 19: 83-86Abstract Full Text PDF PubMed Scopus (87) Google Scholar). The molecular mechanism by which these proteins discriminate a large number of chemically unrelated DNA lesions as substrates for NER is unknown. However, the versatility of NER led to the assumption that this system recognizes conformational changes imposed on DNA at sites of damage rather than specific base modifications (2Sancar A. J. Biol. Chem. 1995; 270: 15915-15918Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 5Van Houten B. Microbiol. Rev. 1990; 54: 18-51Crossref PubMed Google Scholar). In this report, we compared recognition of DNA adducts where detailed structural information is available and identified a molecular determinant triggering initiation of the mammalian NER pathway.The acetylaminofluorene (AAF), benzo[a]pyrene diol-epoxide (BPDE), 8-methoxypsoralen (8-MOP), anthramycin, and CC-1065 moieties are illustrated in Fig. 1. Melting temperature studies have shown that these bulky base adducts alter the thermodynamic characteristics of DNA in different ways. AAF adducts (14O'Handley S.F. Sanford S.G. Xu R. Lester C.C. Hingerty B.E. Broyde S. Krugh T.R. Biochemistry. 1993; 32: 2481-2497Crossref PubMed Scopus (141) Google Scholar, 15Garcia A. Lambert I.B. Fuchs R.P.P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5989-5993Crossref PubMed Scopus (80) Google Scholar), BPDE adducts (16Zou Y. Liu T.-M. Geacintov N.E. Van Houten B. Biochemistry. 1995; 34: 13582-13593Crossref PubMed Scopus (77) Google Scholar, 17Xu R. Birke S. Carberry S.E. Geacintov N.E. Swenberg C.E. Harvey R.G. Nucleic Acids Res. 1992; 20: 6167-6176Crossref PubMed Scopus (22) Google Scholar) and UV radiation products (18Rahn R.O. Patrick M.H. Wang S.Y. Photochemistry and Photobiology of Nucleic Acids. Vol 2. Academic Press, NY1976: 97Google Scholar) destabilize the DNA double helix relative to nonmodified DNA, whereas 8-MOP (19Shi Y.-B. Hearst J.E. Biochemistry. 1986; 25: 5895-5902Crossref PubMed Scopus (55) Google Scholar, 20Shi Y.-B Griffith J. Hearst J.E. Nucleic Acids Res. 1988; 16: 8945-8952Crossref PubMed Scopus (16) Google Scholar), anthramycin (21Hurley L.H. Thurston D.E. Pharmacol. Res. 1984; 1: 53-59Crossref Scopus (61) Google Scholar), and CC-1065 adducts (22Reynolds V.L. McGovren J.P. Hurley L.H. J. Antibiot. (Tokyo). 1986; 39: 319-334Crossref PubMed Scopus (154) Google Scholar) stabilize the DNA duplex. In the predominant adduct formed by N-acetoxy-2-acetylaminofluorene (AAF-C8-guanine), the modified base is rotated out of the helix axis, and the duplex is locally denatured (14O'Handley S.F. Sanford S.G. Xu R. Lester C.C. Hingerty B.E. Broyde S. Krugh T.R. Biochemistry. 1993; 32: 2481-2497Crossref PubMed Scopus (141) Google Scholar, 23Schwartz A. Marrot L. Leng M. J. Mol. Biol. 1989; 207: 445-450Crossref PubMed Scopus (22) Google Scholar). Reaction of anti-7,8-diol 9,10-epoxy-benzo[a]pyrene with double-stranded DNA generates mainly (+)-trans-anti-BPDE-N2-guanine adducts with quantitatively minor lesions resulting from (-)-trans, (+)-cis, and (-)-cis additions to the same position N2 of guanine (24de los Santos C. Cosman M. Hingerty B.E. Ibanez V. Margulis L.A. Geacintov N.E. Broyde S. Patel D.J. Biochemistry. 1992; 31: 5245-5252Crossref PubMed Scopus (185) Google Scholar, 25Cosman M. de los Santos C. Fiala R. Hingerty B.E. Ibanez V. Luna E. Harvey R. Geacintov N.E. Broyde S. Patel D.J. Biochemistry. 1993; 32: 4145-4155Crossref PubMed Scopus (164) Google Scholar). Depending on their stereochemistry, these BPDE-N2-guanine adducts are either accommodated in the minor groove (24de los Santos C. Cosman M. Hingerty B.E. Ibanez V. Margulis L.A. Geacintov N.E. Broyde S. Patel D.J. Biochemistry. 1992; 31: 5245-5252Crossref PubMed Scopus (185) Google Scholar) and cause DNA unwinding (17Xu R. Birke S. Carberry S.E. Geacintov N.E. Swenberg C.E. Harvey R.G. Nucleic Acids Res. 1992; 20: 6167-6176Crossref PubMed Scopus (22) Google Scholar) or assume a base-displacement configuration with localized base pair disruption (25Cosman M. de los Santos C. Fiala R. Hingerty B.E. Ibanez V. Luna E. Harvey R. Geacintov N.E. Broyde S. Patel D.J. Biochemistry. 1993; 32: 4145-4155Crossref PubMed Scopus (164) Google Scholar). For comparison, we also generated helix-destabilizing modifications by irradiating DNA with UV light at 254 nm, producing as major lesions cyclobutane pyrimidine dimers and pyrimidine(6-4) photoproducts in a ratio of about 3:1 (26Cadet J. Anselmino C. Douki T. Voituriez L. J. Photochem. Photobiol. B Biol. 1992; 15: 277-298Crossref PubMed Scopus (184) Google Scholar).Treatment of DNA with 8-MOP and long wavelength UV light (>320 nm) yields psoralen monoadducts and a small proportion of psoralen diadducts (27Hearst J.E. Isaacs S.T. Kanne D. Rapoport H. Straub K. Q. Rev. Biophys. 1984; 17: 1-44Crossref PubMed Scopus (137) Google Scholar). This photoaddition reaction occurs between the 5,6 double bond of pyrimidine bases and either the 3,4 (pyrone) or the 4ʹ,5ʹ (furan) double bond of the psoralen. Modifications with psoralen induce helical distortion by unwinding the duplex and enhancing backbone flexibility (28Spielmann H.P. Dwyer T.J. Hearst J.E. Wemmer D.E. Biochemistry. 1995; 34: 12937-12953Crossref PubMed Scopus (70) Google Scholar) but fail to destabilize the secondary structure of DNA. On the contrary, thermostability measurements showed that both pyroneside and furanside monoadducts stabilize the helix by mediating stacking interactions between the psoralen moiety and the surrounding base pairs (19Shi Y.-B. Hearst J.E. Biochemistry. 1986; 25: 5895-5902Crossref PubMed Scopus (55) Google Scholar, 20Shi Y.-B Griffith J. Hearst J.E. Nucleic Acids Res. 1988; 16: 8945-8952Crossref PubMed Scopus (16) Google Scholar, 28Spielmann H.P. Dwyer T.J. Hearst J.E. Wemmer D.E. Biochemistry. 1995; 34: 12937-12953Crossref PubMed Scopus (70) Google Scholar). Helix-stabilizing adducts were also obtained using anthramycin, a pyrrolo[1,4]benzodiazepine antibiotic, and CC-1065, a composite compound consisting of three pyrroloindole subunits joined by amide linkages (Fig. 1). Anthramycin binds selectively to N2 of guanine through aminal bonds and forms covalent adducts with essentially no distortion of the DNA helix (21Hurley L.H. Thurston D.E. Pharmacol. Res. 1984; 1: 53-59Crossref Scopus (61) Google Scholar, 29Krugh T.R. Graves D.E. Stone M. Biochemistry. 1989; 28: 9988-9994Crossref PubMed Scopus (32) Google Scholar). CC-1065 displays a cyclopropyl ring that alkylates DNA at position N3 of adenine, generating covalent adducts that cause bending and winding of the double helix (22Reynolds V.L. McGovren J.P. Hurley L.H. J. Antibiot. (Tokyo). 1986; 39: 319-334Crossref PubMed Scopus (154) Google Scholar, 30Lee C.-S. Sun D. Kizu R. Hurley L.H. Chem. Res. Toxicol. 1991; 4: 203-213Crossref PubMed Scopus (56) Google Scholar). Both anthramycin and CC-1065 adducts enhance duplex stability through noncovalent interactions derived from hydrogen bonds (anthramycin) or van der Waals and hydrophobic forces (CC-1065) within the minor groove of DNA (21Hurley L.H. Thurston D.E. Pharmacol. Res. 1984; 1: 53-59Crossref Scopus (61) Google Scholar, 22Reynolds V.L. McGovren J.P. Hurley L.H. J. Antibiot. (Tokyo). 1986; 39: 319-334Crossref PubMed Scopus (154) Google Scholar, 30Lee C.-S. Sun D. Kizu R. Hurley L.H. Chem. Res. Toxicol. 1991; 4: 203-213Crossref PubMed Scopus (56) Google Scholar).Recognition of these bulky adducts was compared by monitoring their capacity to sequester NER factors. To that end, we have developed a competition assay that measures the efficiency by which damaged plasmids compete for NER factors operating on a site-directed substrate (31Hess M.T. Gunz D. Naegeli H. Nucleic Acids Res. 1996; 24: 824-828Crossref PubMed Scopus (21) Google Scholar). As a source of NER activity we exploited a standard soluble extract from human cells (32Manley J.L. Fire A. Samuels M. Sharp P.A. Methods Enzymol. 1983; 101: 568-582Crossref PubMed Scopus (222) Google Scholar, 33Wood R.D. Robins P. Lindahl T. Cell. 1988; 53: 97-106Abstract Full Text PDF PubMed Scopus (380) Google Scholar). Under the conditions used in our study, this cell-free extract does not support chromatin assembly or transcription, thereby eliminating nuclear activities that modulate the intrinsic capacity of NER to recognize DNA damage (34Wood R.D. Coverley D. BioEssays. 1991; 13: 447-453Crossref PubMed Scopus (27) Google Scholar, 35Hanawalt P.C. Science. 1994; 266: 1957-1958Crossref PubMed Scopus (452) Google Scholar). This system revealed >1000-fold differences in the capacity of the tested adducts to sequester human NER factors. Those lesions that destabilize the DNA helix (AAF or BPDE adducts, UV radiation products) were effective competitors. In contrast, those adducts that stabilize the helix (8-MOP, anthramycin, and CC-1065 adducts) displayed minimal competing effects. In parallel, site-specifically placed CC-1065 adducts were unable to detectably stimulate synthesis of DNA repair patches. These results indicate that an early subset of NER recognition factors is attracted to structural defects associated with unfavorable thermodynamic changes of the DNA double helix. As a consequence, human NER is preferentially targeted to sites of helical instability.
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