DNA Repair Excision Nuclease Attacks Undamaged DNA
2001; Elsevier BV; Volume: 276; Issue: 27 Linguagem: Inglês
10.1074/jbc.m101032200
ISSN1083-351X
AutoresMark E. Branum, Joyce T. Reardon, Aziz Sancar,
Tópico(s)DNA and Nucleic Acid Chemistry
ResumoNucleotide excision repair is a general repair system that eliminates many dissimilar lesions from DNA. In an effort to understand substrate determinants of this repair system, we tested DNAs with minor backbone modifications using the ultrasensitive excision assay. We found that a phosphorothioate and a methylphosphonate were excised with low efficiency. Surprisingly, we also found that fragments of 23–28 nucleotides and of 12–13 nucleotides characteristic of human and Escherichia coliexcision repair, respectively, were removed from undamaged DNA at a significant rate. Considering the relative abundance of undamaged DNA in comparison to damaged DNA in the course of the life of an organism, we conclude that, in general, excision from and resynthesis of undamaged DNA may exceed the excision and resynthesis caused by DNA damage. As resynthesis is invariably associated with mutations, we propose that gratuitous repair may be an important source of spontaneous mutations. Nucleotide excision repair is a general repair system that eliminates many dissimilar lesions from DNA. In an effort to understand substrate determinants of this repair system, we tested DNAs with minor backbone modifications using the ultrasensitive excision assay. We found that a phosphorothioate and a methylphosphonate were excised with low efficiency. Surprisingly, we also found that fragments of 23–28 nucleotides and of 12–13 nucleotides characteristic of human and Escherichia coliexcision repair, respectively, were removed from undamaged DNA at a significant rate. Considering the relative abundance of undamaged DNA in comparison to damaged DNA in the course of the life of an organism, we conclude that, in general, excision from and resynthesis of undamaged DNA may exceed the excision and resynthesis caused by DNA damage. As resynthesis is invariably associated with mutations, we propose that gratuitous repair may be an important source of spontaneous mutations. base pair(s) cell-free extract xeroderma pigmentosum excision repair cross-complementing 8-hydroxyguanine nucleotide(s) Nucleotide excision repair is a general repair system that removes damaged bases from DNA by dual incisions of the damaged strand at some distance from the lesion, releasing the damaged base in the form of 12–13-mers in prokaryotes and 24–32-mers in eukaryotes (1Sancar A. Annu. Rev. Biochem. 1996; 65: 43-81Crossref PubMed Scopus (960) Google Scholar, 2Wood R.D. J. Biol. Chem. 1997; 272: 23465-23468Crossref PubMed Scopus (378) Google Scholar). It is the major repair system for bulky base adducts, but it also acts on nonbulky lesions such as oxidized or methylated bases and, as such, functions as a backup system for DNA glycosylases, which have restricted substrate ranges (3Huang 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, 4Reardon J.T. Bessho T. Kung H.C. Bolton P.H. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9463-9468Crossref PubMed Scopus (330) Google Scholar).The wide substrate spectrum of the excision nuclease raises two interrelated questions: what is the substrate range of the enzyme system and how does the enzyme recognize substrate? Both of these questions have been addressed in numerous studies, and at present we have a basic understanding of damage recognition in both prokaryotes and eukaryotes (1Sancar A. Annu. Rev. Biochem. 1996; 65: 43-81Crossref PubMed Scopus (960) Google Scholar, 2Wood R.D. J. Biol. Chem. 1997; 272: 23465-23468Crossref PubMed Scopus (378) Google Scholar, 5Hess M.T. Schwitter U. Petretta M. Giese B. Naegeli H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6664-6669Crossref PubMed Scopus (122) Google Scholar, 6Buschta-Hedayat N. Buterin T. Hess M.T. Missura M. Naegeli H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6090-6095Crossref PubMed Scopus (96) Google Scholar). With regard to substrate range, its limits remain to be defined. The excision nuclease, which originally was thought to be specific for bulky lesions, was later found to excise nonbulky adducts such as methylated bases but, apparently, failed to excise nucleotides with backbone modifications such as the C4′ pivaloyl adduct (5Hess M.T. Schwitter U. Petretta M. Giese B. Naegeli H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6664-6669Crossref PubMed Scopus (122) Google Scholar, 6Buschta-Hedayat N. Buterin T. Hess M.T. Missura M. Naegeli H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6090-6095Crossref PubMed Scopus (96) Google Scholar). With the availability of more efficient in vitro systems (4Reardon J.T. Bessho T. Kung H.C. Bolton P.H. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9463-9468Crossref PubMed Scopus (330) Google Scholar, 7Mu D. Hsu D.S. Sancar A. J. Biol. Chem. 1996; 271: 8285-8294Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 8Matsunaga T. Park C.H. Bessho T. Mu D. Sancar A. J. Biol. Chem. 1996; 271: 11047-11050Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar) we decided to re-examine the question of recognition of backbone modifications. We found that both phosphorothioate and methylphosphonate backbone modifications were recognized as substrates by the human excision nuclease. This, in turn, led us to take a closer look at the effect of the enzyme system on undamaged DNA. We find that both the human and the Escherichia coli excision nucleases excise oligomers of 23–28 and 12–13 nucleotides, respectively, from undamaged DNA. This gratuitous excision and the inevitable repair synthesis that must follow could be potential sources of spontaneous mutations. Our data suggest that even in nondividing cells in which there is no DNA replication, there can be significant DNA turnover due to gratuitous excision and resynthesis and that this gratuitous "repair" may cause mutations in such cells, even when they are protected from all extrinsic and intrinsic DNA damaging agents.DISCUSSIONOur findings raise two questions: why and how is the undamaged DNA attacked by the excision nuclease, and what is the biological role of gratuitous DNA repair? These questions are addressed below.Attack of Excision Nuclease on Undamaged DNAThe precise mechanism of damage recognition by human excision nuclease is not known. Hence, at present, it is not possible to answer the questions of why and how the enzyme attacks undamaged DNA in any detail. Based on the structure of a preincision DNA-enzyme complex, which contains a subset of the repair factors and partially unwound and kinked DNA (1Sancar A. Annu. Rev. Biochem. 1996; 65: 43-81Crossref PubMed Scopus (960) Google Scholar,2Wood R.D. J. Biol. Chem. 1997; 272: 23465-23468Crossref PubMed Scopus (378) Google Scholar, 7Mu D. Hsu D.S. Sancar A. J. Biol. Chem. 1996; 271: 8285-8294Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 13Wakasugi M. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6669-6674Crossref PubMed Scopus (149) Google Scholar), it has been proposed that any DNA structure that is amenable to unwinding and kinking and otherwise assuming the conformation existing in the ultimate preincision complex might function as a substrate (1Sancar A. Annu. Rev. Biochem. 1996; 65: 43-81Crossref PubMed Scopus (960) Google Scholar, 5Hess M.T. Schwitter U. Petretta M. Giese B. Naegeli H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6664-6669Crossref PubMed Scopus (122) Google Scholar, 6Buschta-Hedayat N. Buterin T. Hess M.T. Missura M. Naegeli H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6090-6095Crossref PubMed Scopus (96) Google Scholar). Since even undamaged DNA can assume the conformation of the preincision complex with low but finite probability (1Sancar A. Annu. Rev. Biochem. 1996; 65: 43-81Crossref PubMed Scopus (960) Google Scholar), it is not surprising that undamaged DNA is a substrate for excision nucleases. Indeed, there have been reports on incision of undamaged DNA by the E. coli excinuclease (19Van Houten B. Sancar A. J. Bacteriol. 1987; 169: 540-545Crossref PubMed Google Scholar,20Caron P.R. Grossman L. Nucleic Acids Res. 1988; 16: 7855-7865Crossref PubMed Scopus (32) Google Scholar). In one of those studies, however, uniformly radiolabeled plasmid DNA was used in a nicking assay that is incapable of detecting an excinuclease mode of action (19Van Houten B. Sancar A. J. Bacteriol. 1987; 169: 540-545Crossref PubMed Google Scholar). The second study used linear DNA uniformly labeled with 32P as a substrate in an excision assay, and 9-nt-long oligomers were released instead of the characteristic 12–13-nt-long oligomers (20Caron P.R. Grossman L. Nucleic Acids Res. 1988; 16: 7855-7865Crossref PubMed Scopus (32) Google Scholar). Later work revealed that the 9-mers are released by a potent 3′-exonuclease action of the UvrABC proteins at a nick or a double-strand break (21Gordienko I. Rupp W.D. EMBO J. 1998; 17: 626-633Crossref PubMed Scopus (21) Google Scholar, 22Moolenaar G.F. Bazuine M. van Knippenberg I.C. Visse R. Goosen N. J. Biol. Chem. 1998; 273: 34896-34903Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), and hence the product was not released by an excinuclease type of action (dual incisions in one strand). In this study we present unambiguous evidence that both the human and the E. coli excision nucleases attack undamaged DNA in the typical excinuclease mode.It is very likely that certain DNA sequences would be more susceptible to attack by excision nuclease than others. We have tested three different random sequences and found a similar level of excision by the excision nuclease. A more extensive survey, however, is likely to identify certain sequences and conformations with increased susceptibility to excision nuclease. Indeed, a recent study (23Bacolla A. Jaworski A. Connors T.D. Wells R.D. J. Biol. Chem. 2001; 276: 18597-18604Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) has shown that the poly(purine:pyrimidine) tract present in the polycystic kidney disease gene (PKD1) when present in a supercoiled plasmid is efficiently processed by the E. coliexcinuclease. An extreme case of the effect of DNA conformation on gratuitous repair is the form of gratuitous repair that has been proposed to occur as a side product of transcription-repair coupling (24Hanawalt P.C. Science. 1994; 266: 1957-1958Crossref PubMed Scopus (452) Google Scholar, 25Hanawalt P.C. Mutat. Res. 2001; 485: 3-13Crossref PubMed Scopus (118) Google Scholar). It has been speculated that when RNA polymerase stalls at natural transcriptional pause sites the transcription-coupled repair machinery is activated in a manner similar to RNA polymerase stalling at a lesion and that such activation of the transcription-coupled repair system leads to gratuitous and potentially mutagenic repair. Currently there is no experimental evidence for gratuitous repair initiated by stalled RNA polymerase. However, there are several reports that show that transcribed DNA is mutated at higher frequency than nontranscribed DNA (26Fix D.F. Glickman B.W. Mol. Gen. Genet. 1987; 209: 78-82Crossref PubMed Scopus (52) Google Scholar, 27Davis B.D. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5005-5009Crossref PubMed Scopus (75) Google Scholar, 28Datta A. Jinks-Robertson S. Science. 1995; 268: 1616-1619Crossref PubMed Scopus (212) Google Scholar, 29Beletskii A. Bhagwat A.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13919-13924Crossref PubMed Scopus (192) Google Scholar). Whether this increased mutation frequency is due to transcription-coupled gratuitous DNA repair or the increased susceptibility of single-stranded DNA in the transcription bubble to various DNA damaging agents is not known at present.Biological Relevance of Gratuitous RepairWe suspect that gratuitous excision repair has no beneficial effect for the organism. Removal and replacement of undamaged DNA by nucleotide excision repair is the price the cell has to pay to have an omnipotent DNA repair enzyme capable of handling a virtually infinite variety of lesions. This excision and resynthesis may not be totally innocuous, since it may introduce spontaneous mutations into undamaged DNA as is shown in the following calculation.Fig. 5 compares the relative efficiency of human excision nuclease on a variety of lesions and on undamaged DNA. As is apparent, with the unique substrate and assay system we use, undamaged DNA is excised at a rate of about 1% that of the (6-4) photoproduct, which is the best natural substrate for the enzyme and is used as the "gold standard" for other substrates. However, in calculating the susceptibility of undamaged DNA to excision nuclease activity with the (6-4) photoproduct as a reference, a correction factor must be introduced for the relative abundance of the targets. Essentially all of the excision products from the (6-4) substrate arise from a single lesion, whereas the excision products from undamaged DNA arise from dual incisions over about a 50-nucleotide region in a variety of combinations that bracket the radiolabel (Fig.6). Hence, in calculating the efficiency of the enzyme on an undamaged nucleotide, a correction factor of 50 is introduced, making the actual efficiency of an undamaged base relative to that of a (6-4) photoproduct equal to about 1/(50 × 100) = 2 × 10−4. This might seem insignificant, but if one considers that every nucleotide in the human genome complement is a potential target for attack by the excision nuclease, the level of excision of undamaged DNA becomes significant. The maximum rate of excision of (6-4) photoproducts under substrate saturating condition has been estimated to be 2.7 × 103/min/diploid human cell (30Ye N. Bianchi M.S. Bianchi N.O. Holmquist G.P. Mutat. Res. 1999; 435: 43-61Crossref PubMed Scopus (37) Google Scholar). Assuming that the relative rates we obtained in vitro are applicable to the in vivo environment, it is predicted that every minute (2.7 × 103) × (2 × 10−4) = 5.4 × 10−1 undamaged nucleotides would be subject to excinuclease action, and since each excision event removes about 25 nucleotides, it is calculated that 5.4 × 10−1 × 25 = 13.5 nucleotides/min are removed by the human excision nuclease. This, in turn, means excision and replacement of about 2 × 104 nucleotides per day per human cell. This value is comparable with the nucleotide turnover that occurs under physiological conditions as a result of base excision repair processing of damaged bases (104 to 105per cell per day) arising from depurination, deamination, oxidation, and methylation (31Holmquist G.P. Mutat. Res. 1998; 400: 59-68Crossref PubMed Scopus (53) Google Scholar, 32Kunkel T.A. Bebenek K. Annu. Rev. Biochem. 2000; 69: 497-529Crossref PubMed Scopus (788) Google Scholar). Thus, it is conceivable that gratuitous nucleotide excision repair contributes to DNA turnover as much as base excision repairs acting on spontaneous DNA lesions. Gratuitous repair is not necessarily restricted to the nucleotide excision repair system. It has been shown that certain DNA glycosylases also attack undamaged DNA causing gratuitous repair which, under special conditions, can be mutagenic (33Berdal K.G. Johansen R.F. Seeberg E. EMBO J. 1998; 17: 363-367Crossref PubMed Scopus (156) Google Scholar, 34Wyatt M.D. Allan J.M. Lau A.Y. Ellenberger T.E. Samson L.D. Bioessays. 1999; 21: 668-676Crossref PubMed Scopus (167) Google Scholar). Mismatch repair, like nucleotide excision repair, has a wide substrate range and many mechanistic similarities to nucleotide excision repair (35Modrich P. Lahue R. Annu. Rev. Biochem. 1996; 65: 101-133Crossref PubMed Scopus (1315) Google Scholar, 36Kolodner R. Genes Dev. 1996; 10: 1433-1442Crossref PubMed Scopus (540) Google Scholar) and conceivably may perform gratuitous repair. Since the mismatch repair patches, as a rule, are much larger than those of base or nucleotide excision repair, this system as well may contribute to spontaneous mutagenesis.Figure 6Dual incisions releasing labeled oligonucleotides from undamaged and damaged DNA. The (6-4) photoproduct is released mainly by incisions at the 4thphosphodiester bond 3′ and the 24th phosphodiester bond 5′ to the lesion. With undamaged DNA any combination of incisions about 28 nt apart, bracketing the label, release the appropriate size fragments. The dual incisions representing the extreme locations for releasing the label are shown, and any combination of sites between these two extremes will release the radiolabel from undamaged DNA.View Large Image Figure ViewerDownload (PPT)ConclusionIn this paper we have shown that DNA with minor backbone modifications and nominally unmodified (undamaged) DNA are attacked by the human and E. coli excision nucleases. The concern that the nominally undamaged DNA may in fact contain some cryptic damage can never be unequivocally eliminated. We feel, however, that the excision we observe from undamaged DNA does represent attack on truly undamaged DNA for the following reasons. First, using an analytical probe for the most common spontaneous lesion in DNA, 8-oxoG, we demonstrate that the level of this lesion in our synthetic substrate is well below the level required to account for the level of excision we observe for such undamaged substrate. Second, the fact that even such a minor modification as the replacement of an oxygen by a sulfur in the backbone increases the susceptibility of DNA to the excision nuclease leads to the reasonably logical conclusion that substrate and nonsubstrate DNA are not quantized for the excision nuclease. Instead it suggests that DNA structures ranging from gross distortions to no distortion represent the two extremes of the continuum of excision nuclease substrates. Nucleotide excision repair is a general repair system that removes damaged bases from DNA by dual incisions of the damaged strand at some distance from the lesion, releasing the damaged base in the form of 12–13-mers in prokaryotes and 24–32-mers in eukaryotes (1Sancar A. Annu. Rev. Biochem. 1996; 65: 43-81Crossref PubMed Scopus (960) Google Scholar, 2Wood R.D. J. Biol. Chem. 1997; 272: 23465-23468Crossref PubMed Scopus (378) Google Scholar). It is the major repair system for bulky base adducts, but it also acts on nonbulky lesions such as oxidized or methylated bases and, as such, functions as a backup system for DNA glycosylases, which have restricted substrate ranges (3Huang 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, 4Reardon J.T. Bessho T. Kung H.C. Bolton P.H. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9463-9468Crossref PubMed Scopus (330) Google Scholar). The wide substrate spectrum of the excision nuclease raises two interrelated questions: what is the substrate range of the enzyme system and how does the enzyme recognize substrate? Both of these questions have been addressed in numerous studies, and at present we have a basic understanding of damage recognition in both prokaryotes and eukaryotes (1Sancar A. Annu. Rev. Biochem. 1996; 65: 43-81Crossref PubMed Scopus (960) Google Scholar, 2Wood R.D. J. Biol. Chem. 1997; 272: 23465-23468Crossref PubMed Scopus (378) Google Scholar, 5Hess M.T. Schwitter U. Petretta M. Giese B. Naegeli H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6664-6669Crossref PubMed Scopus (122) Google Scholar, 6Buschta-Hedayat N. Buterin T. Hess M.T. Missura M. Naegeli H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6090-6095Crossref PubMed Scopus (96) Google Scholar). With regard to substrate range, its limits remain to be defined. The excision nuclease, which originally was thought to be specific for bulky lesions, was later found to excise nonbulky adducts such as methylated bases but, apparently, failed to excise nucleotides with backbone modifications such as the C4′ pivaloyl adduct (5Hess M.T. Schwitter U. Petretta M. Giese B. Naegeli H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6664-6669Crossref PubMed Scopus (122) Google Scholar, 6Buschta-Hedayat N. Buterin T. Hess M.T. Missura M. Naegeli H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6090-6095Crossref PubMed Scopus (96) Google Scholar). With the availability of more efficient in vitro systems (4Reardon J.T. Bessho T. Kung H.C. Bolton P.H. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9463-9468Crossref PubMed Scopus (330) Google Scholar, 7Mu D. Hsu D.S. Sancar A. J. Biol. Chem. 1996; 271: 8285-8294Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 8Matsunaga T. Park C.H. Bessho T. Mu D. Sancar A. J. Biol. Chem. 1996; 271: 11047-11050Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar) we decided to re-examine the question of recognition of backbone modifications. We found that both phosphorothioate and methylphosphonate backbone modifications were recognized as substrates by the human excision nuclease. This, in turn, led us to take a closer look at the effect of the enzyme system on undamaged DNA. We find that both the human and the Escherichia coli excision nucleases excise oligomers of 23–28 and 12–13 nucleotides, respectively, from undamaged DNA. This gratuitous excision and the inevitable repair synthesis that must follow could be potential sources of spontaneous mutations. Our data suggest that even in nondividing cells in which there is no DNA replication, there can be significant DNA turnover due to gratuitous excision and resynthesis and that this gratuitous "repair" may cause mutations in such cells, even when they are protected from all extrinsic and intrinsic DNA damaging agents. DISCUSSIONOur findings raise two questions: why and how is the undamaged DNA attacked by the excision nuclease, and what is the biological role of gratuitous DNA repair? These questions are addressed below.Attack of Excision Nuclease on Undamaged DNAThe precise mechanism of damage recognition by human excision nuclease is not known. Hence, at present, it is not possible to answer the questions of why and how the enzyme attacks undamaged DNA in any detail. Based on the structure of a preincision DNA-enzyme complex, which contains a subset of the repair factors and partially unwound and kinked DNA (1Sancar A. Annu. Rev. Biochem. 1996; 65: 43-81Crossref PubMed Scopus (960) Google Scholar,2Wood R.D. J. Biol. Chem. 1997; 272: 23465-23468Crossref PubMed Scopus (378) Google Scholar, 7Mu D. Hsu D.S. Sancar A. J. Biol. Chem. 1996; 271: 8285-8294Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 13Wakasugi M. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6669-6674Crossref PubMed Scopus (149) Google Scholar), it has been proposed that any DNA structure that is amenable to unwinding and kinking and otherwise assuming the conformation existing in the ultimate preincision complex might function as a substrate (1Sancar A. Annu. Rev. Biochem. 1996; 65: 43-81Crossref PubMed Scopus (960) Google Scholar, 5Hess M.T. Schwitter U. Petretta M. Giese B. Naegeli H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6664-6669Crossref PubMed Scopus (122) Google Scholar, 6Buschta-Hedayat N. Buterin T. Hess M.T. Missura M. Naegeli H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6090-6095Crossref PubMed Scopus (96) Google Scholar). Since even undamaged DNA can assume the conformation of the preincision complex with low but finite probability (1Sancar A. Annu. Rev. Biochem. 1996; 65: 43-81Crossref PubMed Scopus (960) Google Scholar), it is not surprising that undamaged DNA is a substrate for excision nucleases. Indeed, there have been reports on incision of undamaged DNA by the E. coli excinuclease (19Van Houten B. Sancar A. J. Bacteriol. 1987; 169: 540-545Crossref PubMed Google Scholar,20Caron P.R. Grossman L. Nucleic Acids Res. 1988; 16: 7855-7865Crossref PubMed Scopus (32) Google Scholar). In one of those studies, however, uniformly radiolabeled plasmid DNA was used in a nicking assay that is incapable of detecting an excinuclease mode of action (19Van Houten B. Sancar A. J. Bacteriol. 1987; 169: 540-545Crossref PubMed Google Scholar). The second study used linear DNA uniformly labeled with 32P as a substrate in an excision assay, and 9-nt-long oligomers were released instead of the characteristic 12–13-nt-long oligomers (20Caron P.R. Grossman L. Nucleic Acids Res. 1988; 16: 7855-7865Crossref PubMed Scopus (32) Google Scholar). Later work revealed that the 9-mers are released by a potent 3′-exonuclease action of the UvrABC proteins at a nick or a double-strand break (21Gordienko I. Rupp W.D. EMBO J. 1998; 17: 626-633Crossref PubMed Scopus (21) Google Scholar, 22Moolenaar G.F. Bazuine M. van Knippenberg I.C. Visse R. Goosen N. J. Biol. Chem. 1998; 273: 34896-34903Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), and hence the product was not released by an excinuclease type of action (dual incisions in one strand). In this study we present unambiguous evidence that both the human and the E. coli excision nucleases attack undamaged DNA in the typical excinuclease mode.It is very likely that certain DNA sequences would be more susceptible to attack by excision nuclease than others. We have tested three different random sequences and found a similar level of excision by the excision nuclease. A more extensive survey, however, is likely to identify certain sequences and conformations with increased susceptibility to excision nuclease. Indeed, a recent study (23Bacolla A. Jaworski A. Connors T.D. Wells R.D. J. Biol. Chem. 2001; 276: 18597-18604Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) has shown that the poly(purine:pyrimidine) tract present in the polycystic kidney disease gene (PKD1) when present in a supercoiled plasmid is efficiently processed by the E. coliexcinuclease. An extreme case of the effect of DNA conformation on gratuitous repair is the form of gratuitous repair that has been proposed to occur as a side product of transcription-repair coupling (24Hanawalt P.C. Science. 1994; 266: 1957-1958Crossref PubMed Scopus (452) Google Scholar, 25Hanawalt P.C. Mutat. Res. 2001; 485: 3-13Crossref PubMed Scopus (118) Google Scholar). It has been speculated that when RNA polymerase stalls at natural transcriptional pause sites the transcription-coupled repair machinery is activated in a manner similar to RNA polymerase stalling at a lesion and that such activation of the transcription-coupled repair system leads to gratuitous and potentially mutagenic repair. Currently there is no experimental evidence for gratuitous repair initiated by stalled RNA polymerase. However, there are several reports that show that transcribed DNA is mutated at higher frequency than nontranscribed DNA (26Fix D.F. Glickman B.W. Mol. Gen. Genet. 1987; 209: 78-82Crossref PubMed Scopus (52) Google Scholar, 27Davis B.D. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5005-5009Crossref PubMed Scopus (75) Google Scholar, 28Datta A. Jinks-Robertson S. Science. 1995; 268: 1616-1619Crossref PubMed Scopus (212) Google Scholar, 29Beletskii A. Bhagwat A.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13919-13924Crossref PubMed Scopus (192) Google Scholar). Whether this increased mutation frequency is due to transcription-coupled gratuitous DNA repair or the increased susceptibility of single-stranded DNA in the transcription bubble to various DNA damaging agents is not known at present.Biological Relevance of Gratuitous RepairWe suspect that gratuitous excision repair has no beneficial effect for the organism. Removal and replacement of undamaged DNA by nucleotide excision repair is the price the cell has to pay to have an omnipotent DNA repair enzyme capable of handling a virtually infinite variety of lesions. This excision and resynthesis may not be totally innocuous, since it may introduce spontaneous mutations into undamaged DNA as is shown in the following calculation.Fig. 5 compares the relative efficiency of human excision nuclease on a variety of lesions and on undamaged DNA. As is apparent, with the unique substrate and assay system we use, undamaged DNA is excised at a rate of about 1% that of the (6-4) photoproduct, which is the best natural substrate for the enzyme and is used as the "gold standard" for other substrates. However, in calculating the susceptibility of undamaged DNA to excision nuclease activity with the (6-4) photoproduct as a reference, a correction factor must be introduced for the relative abundance of the targets. Essentially all of the excision products from the (6-4) substrate arise from a single lesion, whereas the excision products from undamaged DNA arise from dual incisions over about a 50-nucleotide region in a variety of combinations that bracket the radiolabel (Fig.6). Hence, in calculating the efficiency of the enzyme on an undamaged nucleotide, a correction factor of 50 is introduced, making the actual efficiency of an undamaged base relative to that of a (6-4) photoproduct equal to about 1/(50 × 100) = 2 × 10−4. This might seem insignificant, but if one considers that every nucleotide in the human genome complement is a potential target for attack by the excision nuclease, the level of excision of undamaged DNA becomes significant. The maximum rate of excision of (6-4) photoproducts under substrate saturating condition has been estimated to be 2.7 × 103/min/diploid human cell (30Ye N. Bianchi M.S. Bianchi N.O. Holmquist G.P. Mutat. Res. 1999; 435: 43-61Crossref PubMed Scopus (37) Google Scholar). Assuming that the relative rates we obtained in vitro are applicable to the in vivo environment, it is predicted that every minute (2.7 × 103) × (2 × 10−4) = 5.4 × 10−1 undamaged nucleotides would be subject to excinuclease action, and since each excision event removes about 25 nucleotides, it is calculated that 5.4 × 10−1 × 25 = 13.5 nucleotides/min are removed by the human excision nuclease. This, in turn, means e
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