Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Fidelity of Uracil-initiated Base Excision DNA Repair inEscherichia coli Cell Extracts

2001; Elsevier BV; Volume: 276; Issue: 3 Linguagem: Inglês

10.1074/jbc.m008147200

ISSN

1083-351X

Autores

Jung‐Suk Sung, Samuel Bennett, Dale W. Mosbaugh,

Tópico(s)

Bacterial Genetics and Biotechnology

Resumo

The error frequency and mutational specificity associated with Escherichia coli uracil-initiated base excision repair were measured using an M13mp2 lacZαDNA-based reversion assay. Repair was detected in cell-free extracts utilizing a form I DNA substrate containing a site-specific uracil residue. The rate and extent of complete uracil-DNA repair were measured using uracil-DNA glycosylase (Ung)- or double-strand uracil-DNA glycosylase (Dug)-proficient and -deficient isogenicE. coli cells. In reactions utilizing E. coliNR8051 (ung + dug +), ∼80% of the uracil-DNA was repaired, whereas about 20% repair was observed using NR8052 (ung − dug +) cells. The Ung-deficient reaction was insensitive to inhibition by the PBS2 uracil-DNA glycosylase inhibitor protein, implying the involvement of Dug activity. Under both conditions, repaired form I DNA accumulated in conjunction with limited DNA synthesis associated with a repair patch size of 1–20 nucleotides. Reactions conducted with E. coli BH156 (ung − dug +), BH157 (ung + dug −), and BH158 (ung− dug −) cells provided direct evidence for the involvement of Dug in uracil-DNA repair. The rate of repair was 5-fold greater in the Ung-proficient than in the Ung-deficient reactions, while repair was not detected in reactions deficient in both Ung and Dug. The base substitution reversion frequency associated with uracil-DNA repair was determined to be ∼5.5 × 10−4 with transversion mutations dominating the mutational spectrum. In the presence of Dug, inactivation of Ung resulted in up to a 7.3-fold increase in mutation frequency without a dramatic change in mutational specificity. The error frequency and mutational specificity associated with Escherichia coli uracil-initiated base excision repair were measured using an M13mp2 lacZαDNA-based reversion assay. Repair was detected in cell-free extracts utilizing a form I DNA substrate containing a site-specific uracil residue. The rate and extent of complete uracil-DNA repair were measured using uracil-DNA glycosylase (Ung)- or double-strand uracil-DNA glycosylase (Dug)-proficient and -deficient isogenicE. coli cells. In reactions utilizing E. coliNR8051 (ung + dug +), ∼80% of the uracil-DNA was repaired, whereas about 20% repair was observed using NR8052 (ung − dug +) cells. The Ung-deficient reaction was insensitive to inhibition by the PBS2 uracil-DNA glycosylase inhibitor protein, implying the involvement of Dug activity. Under both conditions, repaired form I DNA accumulated in conjunction with limited DNA synthesis associated with a repair patch size of 1–20 nucleotides. Reactions conducted with E. coli BH156 (ung − dug +), BH157 (ung + dug −), and BH158 (ung− dug −) cells provided direct evidence for the involvement of Dug in uracil-DNA repair. The rate of repair was 5-fold greater in the Ung-proficient than in the Ung-deficient reactions, while repair was not detected in reactions deficient in both Ung and Dug. The base substitution reversion frequency associated with uracil-DNA repair was determined to be ∼5.5 × 10−4 with transversion mutations dominating the mutational spectrum. In the presence of Dug, inactivation of Ung resulted in up to a 7.3-fold increase in mutation frequency without a dramatic change in mutational specificity. base excision repair apurinic/apyrimidinic deoxyribose 5′-phosphate polymerase chain reaction base pair(s) 2′-deoxyribonucleoside α- thiotriphosphate Uracil-mediated base excision DNA repair serves as a strategic cellular defense mechanism to maintain the genetic stability of theEscherichia coli genome (1Mosbaugh D.W. Bennett S.E. Prog. Nucleic Acids Res. Mol. Biol. 1994; 48: 315-370Crossref PubMed Scopus (97) Google Scholar). This BER1 process protects the DNA from premutagenic U·G 2Base pairs and mispairs are described by listing the base in the repaired strand first and then the base in the template strand. 2Base pairs and mispairs are described by listing the base in the repaired strand first and then the base in the template strand.mispairs formed by cytosine deamination and U·A base pairs produced by incorporation of dUMP during DNA synthesis (2Duncan B.K. Miller J.H. Nature. 1980; 287: 560-561Crossref PubMed Scopus (556) Google Scholar, 3Tye B.-K. Chien J. Lehman I.R. Duncan B.K. Warner H.R. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 233-237Crossref PubMed Scopus (111) Google Scholar). BER is initiated when uracil-DNA glycosylase recognizes a uracil residue in DNA and catalyzes the cleavage of the N-glycosylic bond that links the uracil base to the deoxyribose phosphate DNA backbone (4Lindahl T. Ljungquist S. Siegert W. Nyberg B. Sperens B. J. Biol. Chem. 1977; 252: 3286-3294Abstract Full Text PDF PubMed Google Scholar). This hydrolytic reaction results in the release of free uracil and creates an abasic site in the DNA (4Lindahl T. Ljungquist S. Siegert W. Nyberg B. Sperens B. J. Biol. Chem. 1977; 252: 3286-3294Abstract Full Text PDF PubMed Google Scholar). Incision by a class II AP endonuclease, either endonuclease IV (Nfo) (5Chan E. Weiss B. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3189-3193Crossref PubMed Scopus (137) Google Scholar) or exonuclease III (Xth) (6Gossard F. Verly W.G. Eur. J. Biochem. 1978; 82: 321-332Crossref PubMed Scopus (59) Google Scholar), cleaves the phosphodiester bond on the 5′-side of the AP site to generate a terminal 3′-hydroxyl-containing nucleotide and a deoxyribose 5′-phosphate residue (7Levin J.D. Demple B. Nucleic Acids Res. 1990; 18: 5069-5075Crossref PubMed Scopus (68) Google Scholar). Approximately 90% of the AP endonuclease activity detected in E. coli is accounted for by the Xth protein (8Doetsch P.W. Cunningham R.P. Mutation Res. 1990; 236: 173-201Crossref PubMed Scopus (322) Google Scholar). Removal of the dRP moiety prior to gap-filling DNA synthesis can occur by the deoxyribophosphodiesterase activity of RecJ, which also possesses a 5′ to 3′ single-stranded DNA exonuclease (9Franklin W.A. Lindahl T. EMBO J. 1988; 7: 3617-3622Crossref PubMed Scopus (60) Google Scholar,10Dianov G. Sedgwick B. Daly G. Olsson M. Lovett S. Lindahl T. Nucleic Acids Res. 1994; 22: 993-998Crossref PubMed Scopus (86) Google Scholar). In addition, the product of the mutM gene (Fpg) has been reported to catalyze the 5′ to 3′ removal of incised dRP residues by a β-elimination mechanism (11Graves R.J. Felzenszwalb I. Laval J. O'Connor T.R. J. Biol. Chem. 1992; 267: 14429-14435Abstract Full Text PDF PubMed Google Scholar). Furthermore, exonuclease I (SbcB) has been shown to release dRP from an AP site incised by Nfo and also to remove 4-hydroxy-2-pentenal-5-phosphate from an abasic site incised by AP lyase on the 3′-side of the lesion (12Sandigursky M. Franklin W.A. Nucleic Acids Res. 1992; 20: 4699-4703Crossref PubMed Scopus (39) Google Scholar). Collectively, RecJ, Fpg, and SbcB may be responsible for dRP removal in E. coli, since inactivation of all three dRP excision activities produced a lethal phenotype (12Sandigursky M. Franklin W.A. Nucleic Acids Res. 1992; 20: 4699-4703Crossref PubMed Scopus (39) Google Scholar). In association with the dRP excision step, one or more nucleotides may be removed from the uracil-containing DNA strand by a 5′ to 3′ exonuclease activity. However, appreciable degradation of the incision site in the 3′ to 5′ direction does not seem to occur (13Dianov G. Price A. Lindahl T. Mol. Cell. Biol. 1992; 12: 1605-1612Crossref PubMed Scopus (259) Google Scholar). Gap-filling DNA synthesis replaces the excised nucleotide(s) by the action of a DNA polymerase, and DNA ligase re-establishes the continuity of the phosphodiester backbone (10Dianov G. Sedgwick B. Daly G. Olsson M. Lovett S. Lindahl T. Nucleic Acids Res. 1994; 22: 993-998Crossref PubMed Scopus (86) Google Scholar).In E. coli, two genetically distinct forms of uracil-DNA glycosylase have been purified to apparent homogeneity, characterized, and demonstrated to initiate uracil-mediated BER (4Lindahl T. Ljungquist S. Siegert W. Nyberg B. Sperens B. J. Biol. Chem. 1977; 252: 3286-3294Abstract Full Text PDF PubMed Google Scholar, 14Sung J.-S. Mosbaugh D.W. Biochemistry. 2000; 39: 10224-10235Crossref PubMed Scopus (40) Google Scholar, 15Varshney U. Hutcheon T. van de Sande J.H. J. Biol. Chem. 1988; 263: 7776-7784Abstract Full Text PDF PubMed Google Scholar, 16Saparbaev M. Laval J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8508-8513Crossref PubMed Scopus (163) Google Scholar, 17Barrett T.E. Savva R. Panayotou G. Barlow T. Brown T. Jiricny J. Pearl L.H. Cell. 1998; 92: 117-129Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). E. coli uracil-DNA glycosylase (Ung) was the first DNA glycosylase identified and consists of a monofunctional single polypeptide with a molecular mass of 25,558 daltons (18Roy S. Purnapatre K. Handa P. Boyanapalli M. Varshney U. Protein Expression Purif. 1998; 13: 155-162Crossref PubMed Scopus (17) Google Scholar, 19Bennett S.E. Jensen O.N. Barofsky D.F. Mosbaugh D.W. J. Biol. Chem. 1994; 269: 21870-21879Abstract Full Text PDF PubMed Google Scholar). Ung prefers to act on uracil residues located in single-stranded DNA but also recognizes uracil in duplex DNA with moderately reduced efficiency (20Bennett S.E. Sanderson R.J. Mosbaugh D.W. Biochemistry. 1995; 34: 6109-6119Crossref PubMed Scopus (82) Google Scholar). A second protein, referred to as double-stranded uracil-DNA glycosylase (Dug, also termed Mug), was recently purified as a 18,672 molecular weight polypeptide and characterized (14Sung J.-S. Mosbaugh D.W. Biochemistry. 2000; 39: 10224-10235Crossref PubMed Scopus (40) Google Scholar, 16Saparbaev M. Laval J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8508-8513Crossref PubMed Scopus (163) Google Scholar). Based on amino acid sequence alignment, Dug lacks strong sequence identity (∼10%) with Ung but shares remarkable similarity in tertiary structure (17Barrett T.E. Savva R. Panayotou G. Barlow T. Brown T. Jiricny J. Pearl L.H. Cell. 1998; 92: 117-129Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Dug preferentially removes uracil from DNA containing a U·G or U·T mispair but inefficiently recognizes a U·A base pair target and lacks detectable activity on single-stranded uracil-containing DNA (14Sung J.-S. Mosbaugh D.W. Biochemistry. 2000; 39: 10224-10235Crossref PubMed Scopus (40) Google Scholar,16Saparbaev M. Laval J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8508-8513Crossref PubMed Scopus (163) Google Scholar).The activity of Ung can also be differentiated from that of Dug based on several other biochemical properties: (i) Ung activity is inhibited by the PBS-1 and -2 uracil-DNA glycosylase inhibitor (Ugi) protein, which forms an essentially irreversible Ung·Ugi complex (21Bennett S.E. Mosbaugh D.W. J. Biol. Chem. 1992; 267: 22512-22521Abstract Full Text PDF PubMed Google Scholar), whereas Dug activity is insensitive to Ugi (14Sung J.-S. Mosbaugh D.W. Biochemistry. 2000; 39: 10224-10235Crossref PubMed Scopus (40) Google Scholar, 17Barrett T.E. Savva R. Panayotou G. Barlow T. Brown T. Jiricny J. Pearl L.H. Cell. 1998; 92: 117-129Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 22Gallinari P. Jiricny J. Nature. 1996; 383: 735-738Crossref PubMed Scopus (183) Google Scholar); (ii) purified Dug is a relatively inefficient enzyme that exhibits a significantly lower uracil excision turnover number than Ung (4Lindahl T. Ljungquist S. Siegert W. Nyberg B. Sperens B. J. Biol. Chem. 1977; 252: 3286-3294Abstract Full Text PDF PubMed Google Scholar, 14Sung J.-S. Mosbaugh D.W. Biochemistry. 2000; 39: 10224-10235Crossref PubMed Scopus (40) Google Scholar, 17Barrett T.E. Savva R. Panayotou G. Barlow T. Brown T. Jiricny J. Pearl L.H. Cell. 1998; 92: 117-129Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar); (iii) Dug is strongly inhibited by apyrimidinic sites but not by free uracil, whereas Ung exhibits modest inhibition by both reaction end products (14Sung J.-S. Mosbaugh D.W. Biochemistry. 2000; 39: 10224-10235Crossref PubMed Scopus (40) Google Scholar); and (iv) Dug efficiently cleaves 3,N 4-ethenocytosine from duplex DNA, while this exocyclic DNA adduct remains refractory to removal by Ung (14Sung J.-S. Mosbaugh D.W. Biochemistry. 2000; 39: 10224-10235Crossref PubMed Scopus (40) Google Scholar, 16Saparbaev M. Laval J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8508-8513Crossref PubMed Scopus (163) Google Scholar). The involvement of Ung in uracil-DNA repair has been well documented (1Mosbaugh D.W. Bennett S.E. Prog. Nucleic Acids Res. Mol. Biol. 1994; 48: 315-370Crossref PubMed Scopus (97) Google Scholar,13Dianov G. Price A. Lindahl T. Mol. Cell. Biol. 1992; 12: 1605-1612Crossref PubMed Scopus (259) Google Scholar, 23Sandigursky M. Freyer G.A. Franklin W.A. Nucleic Acids Res. 1998; 26: 1282-1287Crossref PubMed Scopus (18) Google Scholar); however, the role of Dug remains to be fully elucidated. Sung and Mosbaugh (14Sung J.-S. Mosbaugh D.W. Biochemistry. 2000; 39: 10224-10235Crossref PubMed Scopus (40) Google Scholar) recently demonstrated the involvement of Dug in BER using an E. coli NR8052 (ung) cell-free extract and an M13mp2 form I DNA substrate containing a site-specific U·G mispair. Uracil-initiated BER conducted in the absence of Ung was shown to be both insensitive to Ugi and stimulated by the addition of exogenous Dug (14Sung J.-S. Mosbaugh D.W. Biochemistry. 2000; 39: 10224-10235Crossref PubMed Scopus (40) Google Scholar). Thus, while it has been established that E. coli possesses two classes of uracil-DNA glycosylase capable of initiating BER, the relative contribution of Ung and Dug in mediating repair remains to be determined.Studies of the repair patch size associated with gap-filling DNA synthesis in conjunction with uracil-initiated BER have been conducted using both E. coli cell-free extracts and reconstituted enzyme systems (13Dianov G. Price A. Lindahl T. Mol. Cell. Biol. 1992; 12: 1605-1612Crossref PubMed Scopus (259) Google Scholar, 23Sandigursky M. Freyer G.A. Franklin W.A. Nucleic Acids Res. 1998; 26: 1282-1287Crossref PubMed Scopus (18) Google Scholar, 24Dianov G. Lindahl T. Curr. Biol. 1994; 4: 1069-1076Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Two types of repair patch size have been described involving either the incorporation of a single nucleotide (short patch) or 2–19 nucleotides (long patch). Dianov et al. (13Dianov G. Price A. Lindahl T. Mol. Cell. Biol. 1992; 12: 1605-1612Crossref PubMed Scopus (259) Google Scholar) reported that BER initiated at a U·G target site contained in an oligonucleotide (30-mer) DNA substrate was mainly repaired by a short patch process in wild-type E. coliNH5033 cell extracts. Similar results were obtained using the same DNA substrate and a reconstituted enzyme system composed of Ung, Nfo, RecJ, polymerase I, and ligase (24Dianov G. Lindahl T. Curr. Biol. 1994; 4: 1069-1076Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). In these experiments, short patch BER was largely dependent on the presence of RecJ (24Dianov G. Lindahl T. Curr. Biol. 1994; 4: 1069-1076Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). In sharp contrast, Sandigursky et al. (23Sandigursky M. Freyer G.A. Franklin W.A. Nucleic Acids Res. 1998; 26: 1282-1287Crossref PubMed Scopus (18) Google Scholar) reported that E. coliAB1157 cell extracts did not efficiently support short patch repair when a closed circular DNA containing a U·G mispair was used as substrate. In this case, long patch BER was observed that was not dependent on either SbcB and RecJ or Fpg and RecJ (23Sandigursky M. Freyer G.A. Franklin W.A. Nucleic Acids Res. 1998; 26: 1282-1287Crossref PubMed Scopus (18) Google Scholar). Given the apparent disparity between these two reports, further investigation into the BER patch size is warranted.The biochemical steps involved in E. coli uracil-initiated BER have been elucidated in substantial detail. However, an assessment of the fidelity of DNA repair synthesis associated with the completed BER process initiated by either E. coli Ung or Dug has not been made. In the present study, we have (i) used a site-specific uracil-containing circular duplex DNA substrate to monitor the relative rate of Ung- and Dug-initiated BER in E. coli cell extracts; (ii) determined the repair patch size associated with BER initiated by the two uracil-DNA glycosylases; (iii) measured the base substitution error frequency produced during BER; and (iv) assessed the error specificity associated with Ung- and Dug-mediated BER.DISCUSSIONWe have examined the ability of various E. coli cell extracts to carry out uracil-initiated BER of a covalently closed circular M13mp2 lacZα DNA substrate for the purpose of determining the fidelity associated with complete BER reactions. To our knowledge, this report is the first to present fidelity measurements of BER associated with Ung-proficient or Ung-deficient E. colicells. Several previous studies have implicated Dug as participating in uracil-DNA repair in Ung-deficient cells (14Sung J.-S. Mosbaugh D.W. Biochemistry. 2000; 39: 10224-10235Crossref PubMed Scopus (40) Google Scholar, 16Saparbaev M. Laval J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8508-8513Crossref PubMed Scopus (163) Google Scholar, 22Gallinari P. Jiricny J. Nature. 1996; 383: 735-738Crossref PubMed Scopus (183) Google Scholar); however, a recent report concluded that Dug plays no role in uracil-mediated BER (35Lutsenko E. Bhagwat A.S. J. Biol. Chem. 1999; 274: 31034-31038Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The results presented here support the proposition that Dug can participate in uracil-DNA repair and provide additional evidence that Dug is primarily responsible for uracil-mediated BER in the absence of Ung. These findings reinforce the observations of Gallinari and Jiricny (22Gallinari P. Jiricny J. Nature. 1996; 383: 735-738Crossref PubMed Scopus (183) Google Scholar), who originally identified the double-stranded uracil-DNA glycosylase activity in cell-free extracts of E. coli NR8052 that carried the ung-1 mutation; however, they do not exclude the possibility that Dug may also play a role in the repair of 3,N 4-ethenocytosine residues, as has been suggested by other investigators (16Saparbaev M. Laval J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8508-8513Crossref PubMed Scopus (163) Google Scholar, 35Lutsenko E. Bhagwat A.S. J. Biol. Chem. 1999; 274: 31034-31038Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar).Several lines of evidence support the interpretation that the uracil-mediated repair observed in this study occurred via a BER pathway. First, we observed that upon conclusion of the BER reaction, form I DNA was generated that was resistant to cleavage by the combined treatment of Ung and Nfo. This finding indicated that all steps of uracil-DNA repair had been completed. Second, we demonstrated that DNA synthesis occurred preferentially in the HinfI DNA fragment (529 bp) encompassing the uracil target. Furthermore, DNA synthesis within the 529-bp fragment was almost exclusively dependent on the presence of a uracil residue. Third, the addition of Ugi to cell extracts of E. coli NR8051 substantially inhibited the formation of Ung/Nfo-resistant form I DNA. However, complete inhibition was not observed, since E. coli NR8051 is proficient for Dug activity, which is insensitive to inhibition by Ugi (14Sung J.-S. Mosbaugh D.W. Biochemistry. 2000; 39: 10224-10235Crossref PubMed Scopus (40) Google Scholar). Fourth, DNA synthesis was associated with a repair patch involving ≤20 nucleotides and was oriented 3′ to the uracil target. These results were not characteristic of the E. coli methyl-directed DNA mismatch repair pathway, where DNA repair synthesis tracts of 1 kilobase pair or more can occur (36Lahue R.S. Au K.G. Modrich P. Science. 1989; 245: 160-164Crossref PubMed Scopus (444) Google Scholar). Last, formation of Ung/Nfo-resistant form I DNA was not observed in BER reactions containing extracts of E. coli BH158, in which both ung and dug are inactivated. This observation strongly suggests that Ung and Dug are the predominant, if not exclusive, uracil excision activities in wild type E. coli, and that BER was initiated by one or the other uracil-DNA glycosylase.Examination of the kinetics of BER in extracts of NR8051 (ung +) cells showed that 60% of the M13mp2op14 (U·T) DNA substrate was repaired after a 20-min reaction. In contrast, the rate of BER in extracts of NR8052 (ung−) cells was ∼5.5-fold lower. One interpretation of these data is that Ung rapidly turns over during BER, whereas Dug has a low rate of turnover. Consistent with this interpretation is the observation of Sung and Mosbaugh (14Sung J.-S. Mosbaugh D.W. Biochemistry. 2000; 39: 10224-10235Crossref PubMed Scopus (40) Google Scholar, 17Barrett T.E. Savva R. Panayotou G. Barlow T. Brown T. Jiricny J. Pearl L.H. Cell. 1998; 92: 117-129Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar) that the addition of purified Dug to the Ung-deficient reaction led to an increase in the rate of repair early in the reaction time course. Since strong binding by Dug to its reaction product AP site·G DNA has been demonstrated (14Sung J.-S. Mosbaugh D.W. Biochemistry. 2000; 39: 10224-10235Crossref PubMed Scopus (40) Google Scholar), it is tempting to speculate that Dug binding hinders efficient processing of the abasic site, impeding completion of the BER pathway. While E. coli endonuclease IV was shown to stimulate the catalytic turnover of Dug, Dug-mediated BER remained significantly less than Ung-initiated BER, even under the stimulated condition (14Sung J.-S. Mosbaugh D.W. Biochemistry. 2000; 39: 10224-10235Crossref PubMed Scopus (40) Google Scholar, 17Barrett T.E. Savva R. Panayotou G. Barlow T. Brown T. Jiricny J. Pearl L.H. Cell. 1998; 92: 117-129Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Thus, the role of Dug in uracil-DNA repair conducted via the BER pathway may be to provide an auxiliary repair system that serves as a secondary line of defense against uracil-provoked mutagenesis.What influence does the fidelity of BER have on uracil-initiated mutagenesis in E. coli? Based on the results reported in Table I, the error frequency associated with Ung-mediated BER in cell-free extracts was determined to be 5.5 × 10−4 per repaired uracil residue. Under normal conditions, the vast majority of uracil in E. coli DNA results from dUMP incorporation during replication (1Mosbaugh D.W. Bennett S.E. Prog. Nucleic Acids Res. Mol. Biol. 1994; 48: 315-370Crossref PubMed Scopus (97) Google Scholar, 3Tye B.-K. Chien J. Lehman I.R. Duncan B.K. Warner H.R. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 233-237Crossref PubMed Scopus (111) Google Scholar). Tye et al. (3Tye B.-K. Chien J. Lehman I.R. Duncan B.K. Warner H.R. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 233-237Crossref PubMed Scopus (111) Google Scholar) reported that 1 uracil residue was introduced per 1200 nucleotides polymerized. Accordingly, one would anticipate that ∼4000 uracil residues are incorporated per round of chromosomal DNA replication (1Mosbaugh D.W. Bennett S.E. Prog. Nucleic Acids Res. Mol. Biol. 1994; 48: 315-370Crossref PubMed Scopus (97) Google Scholar). Based on these reports, we extrapolate that E. coli Ung-proficient BER could generate approximately two mutations per cycle of semiconservative DNA replication, providing that error correction did not occur prior to mutation fixation. The addition of Ugi to the Ung-proficient BER reactions resulted in a ∼7-fold increase in reversion frequency without an accompanying change in the mutational specificity. Thus, Ugi produced an ung phenotype that reflected the elevated reversion frequency and mutational specificity associated with Dug-mediated BER. The ungmutator phenotype was reproduced in strains sharing the E. coli GM31 genetic background, namely BH156 and BH157. Taken together, these results indicate that Ung-mediated BER occurs with higher fidelity than that initiated by Dug.The mutational specificity of E. coli uracil-initiated BER repair observed in the opal codon 14 TGA reversion assay appears distinct from that observed in other fidelity assays conducted with purified E. coli DNA polymerase I (large fragment). In extracts of Ung-proficient E. coli cells (NR8051), our mutational analysis revealed that T to G transversions, resulting presumably from T·C mispairs, were dominant (56 of 79), while T to A transversions, the likely result of T·T mispairs, comprised the remainder (23 of 79). In contrast, Minnick et al. (37Minnick D.T. Bebenek K. Osheroff W.P. Turner R.M.J. Astatke M. Liu L. Kunkel T.A. Joyce C.M. J. Biol. Chem. 1999; 274: 3067-3075Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), using a 361-base gap-filling TGA reversion assay and 3′ to 5′ exonuclease-deficient DNA polymerase I (large fragment), observed that the error rate of dGTP incorporation opposite T was ∼49-fold greater than that of dCTP incorporation opposite T and ∼10-fold greater than dTTP incorporation opposite T. Perhaps it is not surprising that our results differ from those reported by Minnick et al. (37Minnick D.T. Bebenek K. Osheroff W.P. Turner R.M.J. Astatke M. Liu L. Kunkel T.A. Joyce C.M. J. Biol. Chem. 1999; 274: 3067-3075Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), since we have utilized a system in which all steps of the BER pathway are represented. The mutational specificity of uracil-mediated BER is the end result of DNA repair synthesis, which includes misincorporation, proofreading, and/or misextension, and must be followed by ligation. On the other hand, the 361-base gap-filling assay is restricted to measurement of the accuracy of the polymerization step in the absence of competing reactions and does not require ligation.We examined the patch size of BER DNA synthesis associated with Ung-versus Dug-mediated repair. In both cases, the patch size was heterogeneous, ranging from 1 to ∼20 nucleotides in length, although the size of the repair patch produced in Dug-mediated BER reactions was consistently somewhat shorter. Quantification of the distribution of the repair patches showed that the mean patch size was 11 nucleotides in Ung-mediated BER compared with 7 nucleotides in Dug-mediated BER. A small amount (∼7%) of 1-nucleotide replacement synthesis was observed in both systems; however, the predominant type of DNA repair synthesis was long patch. The latter observation is consistent with the results of Sandigursky et al. (23Sandigursky M. Freyer G.A. Franklin W.A. Nucleic Acids Res. 1998; 26: 1282-1287Crossref PubMed Scopus (18) Google Scholar), who found that repair of a U·G base pair in a closed circular plasmid involved replacement of ∼15 nucleotides downstream of the uracil target. The first experiments conducted to elucidate the repair patch size associated with BER in E. coli cell extracts utilized a duplex oligodeoxynucleotide 30-mer DNA with a single U·G base pair located approximately in the middle of the substrate (13Dianov G. Price A. Lindahl T. Mol. Cell. Biol. 1992; 12: 1605-1612Crossref PubMed Scopus (259) Google Scholar). Interestingly, under these conditions, more than 70% of DNA repair synthesis involved incorporation of a single nucleotide (13Dianov G. Price A. Lindahl T. Mol. Cell. Biol. 1992; 12: 1605-1612Crossref PubMed Scopus (259) Google Scholar). As previously pointed out, the size of the repair patch may be influenced by the nature of the DNA repair substrate (23Sandigursky M. Freyer G.A. Franklin W.A. Nucleic Acids Res. 1998; 26: 1282-1287Crossref PubMed Scopus (18) Google Scholar, 38Biade S. Sobol R.W. Wilson S.H. Matsumoto Y. J. Biol. Chem. 1998; 273: 898-902Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Thus, short oligodeoxynucleotide substrates may not provide a platform sufficient for interaction with DNA polymerase and accessory repair proteins.It is generally accepted that DNA polymerase I occupies the primary role in uracil-mediated DNA repair synthesis in E. coli (39Tye B.-K. Nyman P.-O. Lehman I.R. Hochhauser S. Weiss B. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 154-157Crossref PubMed Scopus (224) Google Scholar,40Lindahl T. Prog. Nucleic Acids Res. Mol. Biol. 1979; 22: 135-192Crossref PubMed Scopus (431) Google Scholar). Our analysis of the mutational spectra derived from Ung-proficient and Ung-deficient extracts suggests that the specificity of misinsertion remains essentially the same regardless of the uracil-DNA glycosylase involved; accordingly, one might infer that the same DNA polymerase is involved in Ugi-resistant as well as in Ugi-sensitive uracil-DNA repair. Given the relatively high mutation frequencies we observed in this study, a role for the newly discovered polymerase IV and/or polymerase V DNA polymerases in BER cannot be formally excluded. These DNA polymerases have been described as low fidelity enzymes that exhibit error rates of ∼10−3 to 5 × 10−4 when copying undamaged DNA in vitro (41Tang M. Pham P. Shen X. Taylor J.S. O'Donnell M. Woodgate R. Goodman M.F. Nature. 2000; 404: 1014-1018Crossref PubMed Scopus (387) Google Scholar); however, experiments to assess the contribution of these enzymes to BER have not yet been carried out. The molecular mechanisms underlying the mutational specificity of uracil-initiated BER in E. coli and the increased reversion frequency associated with the Dug-mediated BER pathway await further elucidation. Uracil-mediated base excision DNA repair serves as a strategic cellular defense mechanism to maintain the genetic stability of theEscherichia coli genome (1Mosbaugh D.W. Bennett S.E. Prog. Nucleic Acids Res. Mol. Biol. 1994; 48: 315-370Crossref PubMed Scopus (97) Google Scholar). This BER1 process protects the DNA from premut

Referência(s)