Replication Fork Bypass of a Pyrimidine Dimer Blocking Leading Strand DNA Synthesis
1997; Elsevier BV; Volume: 272; Issue: 21 Linguagem: Inglês
10.1074/jbc.272.21.13945
ISSN1083-351X
AutoresMarila Cordeiro‐Stone, Liubov Zaritskaya, Laura K. Price, William K. Kaufmann,
Tópico(s)Epigenetics and DNA Methylation
ResumoWe constructed a double-stranded plasmid containing a single cis,syn-cyclobutane thymine dimer (T[c,s]T) 385 base pairs from the center of the SV40 origin of replication. This circular DNA was replicated in vitro by extracts from several types of human cells. The dimer was placed on the leading strand template of the first replication fork to encounter the lesion. Two-dimensional gel electrophoresis of replication intermediates documented the transient arrest of the replication fork by the dimer. Movement of the replication fork beyond the dimer was recognized by the appearance of a single fork arc in DNA sequences located between the T[c,s]T and the half-way point around the circular template (180° from the origin). Upon completion of plasmid replication, the T[c,s]T was detected by T4 endonuclease V in about one-half (46 ± 9%) of the closed circular daughter molecules. Our results demonstrate that extracts prepared from HeLa cells and SV40-transformed human fibroblasts (SV80, IDH4), including a cell line defective in nucleotide-excision repair (XPA), were competent for leading strand DNA synthesis opposite the pyrimidine dimer and replication fork bypass. In contrast, dimer bypass was severely impaired in otherwise replication-competent extracts from two different xeroderma pigmentosum variant cell lines. We constructed a double-stranded plasmid containing a single cis,syn-cyclobutane thymine dimer (T[c,s]T) 385 base pairs from the center of the SV40 origin of replication. This circular DNA was replicated in vitro by extracts from several types of human cells. The dimer was placed on the leading strand template of the first replication fork to encounter the lesion. Two-dimensional gel electrophoresis of replication intermediates documented the transient arrest of the replication fork by the dimer. Movement of the replication fork beyond the dimer was recognized by the appearance of a single fork arc in DNA sequences located between the T[c,s]T and the half-way point around the circular template (180° from the origin). Upon completion of plasmid replication, the T[c,s]T was detected by T4 endonuclease V in about one-half (46 ± 9%) of the closed circular daughter molecules. Our results demonstrate that extracts prepared from HeLa cells and SV40-transformed human fibroblasts (SV80, IDH4), including a cell line defective in nucleotide-excision repair (XPA), were competent for leading strand DNA synthesis opposite the pyrimidine dimer and replication fork bypass. In contrast, dimer bypass was severely impaired in otherwise replication-competent extracts from two different xeroderma pigmentosum variant cell lines. Solar ultraviolet radiation is a ubiquitous environmental carcinogen responsible for 500,000 or more new cases of skin cancer in the United States each year (1Scotto J. Fears T.R. Fraumeni Jr. J.F. Incidence of Nonmelanoma Skin Cancer in the United States. National Cancer Institute, Bethesda, MD1983: 1-14Google Scholar, 2Ananthaswamy H.N. Pierceall W.E. Photochem. Photobiol. 1990; 52: 1119-1136Google Scholar). Exposure of human cells to natural sunlight leads to the formation of cyclobutane pyrimidine dimers (CPDs), 1The abbreviations used are: CPD, cyclobutane-type pyrimidine dimer; NER, nucleotide-excision repair; PRR, post-replication repair; RFI, closed circular replicative form I; RFII, nicked circular replicative form II; T4 endoV, bacteriophage T4 endonuclease V; Tag, SV40 large T antigen; T[c,s]T,cis,syn-cyclobutane thymine dimer; XPV, xeroderma pigmentosum variant; bp, base pair(s); kb, kilobase pair(s); SF, single fork. pyrimidine(6–4)pyrimidone dimers, and their Dewar valence isomers (3Clingen P.H. Arlett C.F. Roza L. Mori T. Nikaido O. Green M.H.L. Cancer Res. 1995; 55: 2245-2248Google Scholar). UV-induced DNA photoproducts are currently accepted as important underlying factors in skin carcinogenesis (2Ananthaswamy H.N. Pierceall W.E. Photochem. Photobiol. 1990; 52: 1119-1136Google Scholar, 4International Agency for Research on Cancer Working Group IARC Monogr. Eval. Carcinog. Risks Hum. 1992; 55: 43-279Google Scholar). Studies with UVC (254 nm), which forms predominantly CPDs (70–80%) and 6–4 dimers (20–30%), have indicated that mutations and chromosomal aberrations are induced when human cells attempt to replicate the damaged DNA (5Watanabe M. Maher V.M. McCormick J.J. Mutat. Res. 1985; 146: 285-294Google Scholar, 6Kaufmann W.K. Wilson S.J. Mutat. Res. 1994; 314: 67-76Google Scholar). Therefore, the mechanisms whereby human cells complete the replication of template strands containing photoproducts are of considerable interest. UVC inhibits DNA replication in diploid human fibroblast strains by a variety of mechanisms, including G1 arrest (6Kaufmann W.K. Wilson S.J. Mutat. Res. 1994; 314: 67-76Google Scholar) and inhibition of replicon initiation in S phase cells (7Kaufmann W.K. Cleaver J.E. J. Mol. Biol. 1981; 149: 171-187Google Scholar). These two checkpoint responses appear to be protective processes, providing more time for DNA repair to remove photoproducts before DNA replication. UVC also induces inhibition of DNA synthesis in active replicons (7Kaufmann W.K. Cleaver J.E. J. Mol. Biol. 1981; 149: 171-187Google Scholar, 8Boyer J.C. Kaufmann W.K. Brylawski B.P. Cordeiro-Stone M. Cancer Res. 1990; 50: 2593-2598Google Scholar). The latter is thought to reflect, at least in part, the stalling of DNA replication forks at pyrimidine dimers (9Edenberg H.J. Biophys. J. 1976; 16: 849-860Google Scholar, 10Meneghini R. Biochim. Biophys. Acta. 1976; 425: 419-427Google Scholar, 11Meneghini R. Hanawalt P. Biochim. Biophys. Acta. 1976; 425: 428-437Google Scholar), perhaps due to a reduced capacity of DNA polymerases to incorporate DNA precursors into nascent strands opposite template lesions (12Moore P. Strauss B.S. Nature. 1979; 278: 664-666Google Scholar, 13Moore P.D. Bose K.K. Rabkin S.D. Strauss B.S. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 110-114Google Scholar, 14O'Day C.L. Burgers P.M.J. Taylor J.-S. Nucleic Acids Res. 1992; 20: 5403-5406Google Scholar, 15Nelson J.R. Lawrence C.W. Hinckle D.C. Science. 1996; 272: 1646-1649Google Scholar). This inhibition, however, is not absolute, and bypass replication eventually takes place, as evidenced by the generation of replicated DNA containing photoproducts and the induction of point mutations at dipyrimidine sites. UV-induced mutations in the p53 tumor suppressor gene in non-melanoma skin cancers are characterized by a high proportion of C → T transitions (16Ziegler A. Leffell D.J. Kunala S. Sharma H.W. Gailani M. Simon J.A. Halperin A.J. Baden H.P. Shapiro P.E. Bale A.E. Brash D.E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4216-4220Google Scholar, 17Ziegler A. Jonason A.S. Leffell D.J. Simon J.A. Sharma H.W. Kimmelman J. Remington L. Jacks T. Brash D.E. Nature. 1994; 372: 773-776Google Scholar, 18Dumaz N. Stary A. Soussi T. Daya-Grosjean L. Sarasin A. Mutat. Res. 1994; 307: 375-386Google Scholar). Base substitution mutations at thymines do not occur with high frequency in UV-damaged genes that are replicated at their natural chromosomal locations (19McGregor W.G. Chen R.-H. Lukash L. Maher V.M. McCormick J.J. Mol. Cell. Biol. 1991; 11: 1927-1934Google Scholar) or as part of shuttle vectors (20Bredberg A. Kraemer K.H. Seidman M.M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8273-8277Google Scholar, 21Lebkowski J.S. Clancy S. Miller J.H. Calos M.P. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 8606-8610Google Scholar, 22Brash D.E. Seetharam S. Kraemer K.H. Seidman M.M. Bredberg A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3782-3786Google Scholar). Previous studies have demonstrated that protein extracts from HeLa cells are capable of replicating past CPDs during in vitroreplication of UV-damaged plasmids carrying the SV40 origin of replication (23Carty M.P. Hauser J. Levine A.S. Dixon K. Mol. Cell. Biol. 1993; 13: 533-542Google Scholar, 24Thomas D.C. Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7744-7748Google Scholar, 25Svoboda D.L. Vos J.-M.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11975-11979Google Scholar, 26Carty M.P. Lawrence C.W. Dixon K. J. Biol. Chem. 1996; 271: 9637-9647Google Scholar). Experimental evidence in support of this conclusion was found primarily by probing for the presence of sites sensitive to nicking by the CPD-specific enzyme, T4 endonuclease V (T4 endoV), in replicated (DpnI-resistant), closed circular DNA molecules (23Carty M.P. Hauser J. Levine A.S. Dixon K. Mol. Cell. Biol. 1993; 13: 533-542Google Scholar, 24Thomas D.C. Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7744-7748Google Scholar, 25Svoboda D.L. Vos J.-M.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11975-11979Google Scholar, 26Carty M.P. Lawrence C.W. Dixon K. J. Biol. Chem. 1996; 271: 9637-9647Google Scholar). In addition, UV-induced mutagenesis at dipyrimidine sites of randomly damaged plasmids (almost exclusively C → T) presumably reflected error-prone bypass replication (trans-lesion synthesis) of CPDs (23Carty M.P. Hauser J. Levine A.S. Dixon K. Mol. Cell. Biol. 1993; 13: 533-542Google Scholar, 24Thomas D.C. Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7744-7748Google Scholar). Bypass replication of a single dimer strategically placed on one or the other anti-parallel strand of DNA has also been examined (25Svoboda D.L. Vos J.-M.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11975-11979Google Scholar, 26Carty M.P. Lawrence C.W. Dixon K. J. Biol. Chem. 1996; 271: 9637-9647Google Scholar). Inference as to whether bypass replication occurred via leading or lagging strand synthesis was made on the basis of the location and orientation of the dimer, vis à vis the SV40 origin of replication, and the probability of first encounter of the CPD by one or the other of the replication forks moving in opposite direction around the circular molecule. By measuring the relative synthesis of complementary strands at restriction fragments spanning the dimer or located immediately downstream, Svoboda and Vos (25Svoboda D.L. Vos J.-M.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11975-11979Google Scholar) have concluded that when the pyrimidine dimer is on the template to the leading strand the synthesis of the latter is interrupted, but the synthesis of the lagging strand continues, presumably by displacement of the replication fork beyond the lesion. In this study, we have analyzed by two-dimensional gel electrophoresis the topology of replicating molecules to map the displacement of DNA replication forks, in relationship to the position of the CPD, and to demonstrate directly the capability for leading strand bypass replication in human cells. Carty et al. (26Carty M.P. Lawrence C.W. Dixon K. J. Biol. Chem. 1996; 271: 9637-9647Google Scholar) have recently reported that in vitro bypass replication of a single TT site containing either a CPD or a 6–4 dimer is poorly mutagenic. Studies of the familial skin cancer syndrome, xeroderma pigmentosum, have revealed a form of the disease, in which a major biochemical defect in nucleotide excision repair (NER) was not detected, hence the designation of this group as variant (27Jung E.G. Nature. 1970; 228: 361-362Google Scholar, 28Burk P.G. Lutzner M.A. Clarke D.D. Robbins J.H. J. Lab. Clin. Med. 1971; 77: 759-767Google Scholar, 29Cleaver J.E. J. Invest. Dermatol. 1972; 58: 124-128Google Scholar, 30Cleaver J.E. Arutyunyan R.M. Sarkisian T. Kaufmann W.K. Greene A.E. Coriell L. Carcinogenesis. 1980; 1: 647-655Google Scholar, 31Lehmann A.R. Kirk-Bell S. Arlett C.F. Paterson M.C. Lohman P.H.M. de Weerd-Kastelein E.A. Bootsma D. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 219-223Google Scholar). The defining feature of this unique xeroderma pigmentosum group is the abnormal semi-conservative replication of UV-damaged DNA (7Kaufmann W.K. Cleaver J.E. J. Mol. Biol. 1981; 149: 171-187Google Scholar, 8Boyer J.C. Kaufmann W.K. Brylawski B.P. Cordeiro-Stone M. Cancer Res. 1990; 50: 2593-2598Google Scholar, 31Lehmann A.R. Kirk-Bell S. Arlett C.F. Paterson M.C. Lohman P.H.M. de Weerd-Kastelein E.A. Bootsma D. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 219-223Google Scholar, 32Cleaver J.E. Thomas G.H. Park S.D. Biochim. Biophys. Acta. 1979; 564: 122-131Google Scholar, 33Hessel A. Siegle R.J. Mitchell D.L. Cleaver J.E. Arch. Dermatol. 1992; 128: 1233-1237Google Scholar, 34van Zeeland A.A. Filon A.R. Prog. Mutat. Res. 1982; 4: 375-384Google Scholar). The high risk of cancer in sun-exposed skin (35Thielmann H.W. Popanda O. Edler L. Jung E.G. Cancer Res. 1991; 51: 3456-3470Google Scholar, 36Cleaver J.E. Kraemer K.H. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 3. McGraw Hill Inc., New York1995: 4393-4419Google Scholar) and the enhanced sensitivity to UV-induced transformation (37McCormick J.J. Kateley-Kohler S. Watanabe M. Maher V.M. Cancer Res. 1986; 46: 489-492Google Scholar, 38Boyer J.C. Kaufmann W.K. Cordeiro-Stone M. Cancer Res. 1991; 51: 2960-2964Google Scholar) and mutagenesis (39Maher V.M. Ouellette L.M. Curren R.D. McCormick J.J. Nature. 1976; 261: 593-595Google Scholar, 40Myhr B.C. Turnbull D. DiPaolo J.A. Mutat. Res. 1979; 62: 341-353Google Scholar, 41Wang Y.-C. Maher V.M. Mitchell D.L. McCormick J.J. Mol. Cell. Biol. 1993; 13: 4276-4283Google Scholar, 42Waters H.L. Seetharam S. Seidman M.M. Kraemer K.H. J. Invest. Dermatol. 1993; 101: 744-748Google Scholar, 43Raha M. Wang G. Seidman M.M. Glazer P.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2941-2946Google Scholar) support the hypothesis that xeroderma pigmentosum variant (XPV) cells have lost a gene product that participates in an essentially error-free pathway of replication of DNA containing pyrimidine dimers. Our results demonstrate that replication-competent extracts from XPV cells are deficient in the bypass of CPDs, under conditions in which other human cell extracts are capable of catalyzing this process. M13mp2SV was a gift from Dr. Thomas A. Kunkel (NIEHS). Preparations of T4 endoV were provided by Dr. Isabel Mellon (University of Kentucky) and Dr. Stephen Lloyd (University of Texas Medical Branch at Galveston). The oligonucleotide containing the single T[c,s]T dimer was a gift from Dr. Aziz Sancar (University of North Carolina, Chapel Hill). Undamaged oligonucleotides were synthesized by the Nucleic Acids Core Facility of the Lineberger Comprehensive Cancer Center. Eagle's minimal essential medium and l-glutamine were purchased from Life Technologies, Inc. Fetal bovine serum was obtained from HyClone Laboratories Inc. (Logan, UT), and gentamicin came from Elkins-Sinn Inc. (Cherry Hill, NJ). HeLa S3 cells were obtained from the Lineberger Comprehensive Cancer Center Tissue Culture Facility (University of North Carolina, Chapel Hill). Polynucleotide kinase, DNA polymerase, and DNA ligase from bacteriophage T4, as well as restriction enzymes and DNA polymerase I (Klenow fragment used to end label pUC19), were purchased from Boehringer Mannheim. The supplier of purified SV40 large T antigen was Molecular Biology Resources, Inc. (Milwaukee, WI). [α-32P]dCTP (>3000 Ci/mmol) was from Amersham Life Sciences, and unlabeled nucleotides were from Pharmacia Biotech Inc. Creatine phosphate, creatine phosphokinase, and proteinase K were from Sigma. The SV40-transformed cell lines used in this study were derived from XPV fibroblasts, XP4BE (CTag, Ref. 44King S.A. Wilson S.J. Farber R.A. Kaufmann W.K. Cordeiro-Stone M. Exp. Cell Res. 1995; 217: 100-108Google Scholar), and XP30RO (XP30RO/9.8, Ref. 45Volpe J.P.G. Cleaver J.E. Mutat. Res. 1995; 337: 111-117Google Scholar); XPA fibroblasts (XP12BE, GM4429, NIGMS Human Genetic Mutant Cell Repository); and other human fibroblasts (SV80, Ref. 46Choi K.-H. Tevethia S.S. Shin S. Cytogenet. Cell Genet. 1983; 36: 633-640Google Scholar, and IDH4, Ref. 47Shay J.W. West M.D. Wright W.E. Exp. Gerontol. 1992; 27: 477-492Google Scholar). Monolayer cultures were grown in Eagle's minimal essential medium supplemented with 10% fetal bovine serum, 2 mml-glutamine, and 50 μg/ml gentamicin in an atmosphere of 5% CO2 at 37 °C. Extracts were prepared according to published protocols (48Li J.J. Kelly T.J. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 6973-6977Google Scholar, 49Roberts J.D. Kunkel T.A. Methods Mol. Genet. 1993; 2: 295-313Google Scholar). The sequence 5′-GAGCTCAATTAGTCAGCTGC-3′ was introduced into the lacZα sequence of M13mp2SV (50Roberts J.D. Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7064-7068Google Scholar) by site-directed mutagenesis (51Kunkel T.A. Bebenek K. McClary J. Methods Enzymol. 1991; 204: 125-139Google Scholar). This insertion created a unique XhoI and an additional PvuII recognition site in this new construct (M13leaSV, see Fig. 1). Double-stranded, closed circular DNA molecules containing a single pyrimidine dimer were synthesized by annealing closed circular, single-stranded M13leaSV DNA (+ strand), with the oligonucleotide 3′-CTCGAG(T[c,s]T)AATCAGTCGACG-5′, previously phosphorylated at the 5′-end using T4 polynucleotide kinase. This was followed by second-strand synthesis, ligation, and purification in CsCl density gradients, according to published procedures (52Huang J.-C. Svoboda D.L. Reardon J.T. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3664-3668Google Scholar). Undamaged DNA controls included RFI of M13leaSV or molecules prepared as described above but using an oligonucleotide without the dimer. Reactions of 25 or 50 μl contained 30 mm Hepes, pH 7.8, 7 mmMgCl2, 4 mm ATP, 200 μm each of the other three rNTPs, 100 μm each of the four dNTPs, 100–150 μCi/ml [α-32P]dCTP, 40 mmcreatine phosphate, 100 μg/ml creatine phosphokinase, 15 mm sodium phosphate, pH 7.5, 1.6 μg/ml M13leaSV DNA, 40 μg/ml SV40 large T antigen (Tag), 4 mg/ml proteins from human cell extracts (49Roberts J.D. Kunkel T.A. Methods Mol. Genet. 1993; 2: 295-313Google Scholar). Reaction mixtures without Tag were used as negative controls. After incubation at 37 °C for different periods, the reactions were terminated by adding an equal volume of stop solution containing 2% SDS, 2 mg/ml proteinase K, and 50 mm EDTA. In those experiments in which the goal was to quantify specific DNA products fractionated by single dimension agarose gel electrophoresis, an identical amount of linear pUC19 DNA (end-labeled with [α-32P]dCTP by in vitro polymerization at the HindIII cut site) was also added to each reaction. Replication products were purified by one or two extractions with an equal volume of a 1:1 (v/v) mixture of phenol and chloroform/isoamyl alcohol (24:1 v/v), followed by a final extraction with chloroform/isoamyl alcohol and then ethanol precipitation. DNA was routinely dissolved in 10 mm Tris, 1 mm EDTA, pH 7.5, and fractionated in 1% agarose gels containing 0.2 μg/ml ethidium bromide. Dried gels were analyzed with an AMBIS Image Acquisition & Analysis system (AMBIS, Inc., San Diego, CA) or exposed to a phosphor screen that was later scanned by a PhosphorImager™ (Molecular Dynamics, Sunnyvale, CA). DNA replication products from either control or T[c,s]T dimer-containing templates were purified as above and incubated in the presence or absence of T4 endoV. The enzyme preparation received from Dr. Isabel Mellon (485 μg/ml; 2.7 × 1010 nicks/min/μl) was used without dilution at 1 μl per 20-μl reaction containing in vitro replication products in 100 mm NaCl, 10 mm EDTA, 10 mm Tris-HCl, pH 8.0, and 1 mg/ml bovine serum albumin. These reactions were incubated for 1 h at 37 °C. The T4 endoV received from Dr. Stephen Lloyd (stock solution at ∼150 μg/ml) was first diluted 250 or 1000 times in reaction buffer. DNA replication products in 25 mm NaH2PO4, pH 6.8, 1 mm EDTA, 100 mm NaCl, 100 μg/ml bovine serum albumin were mixed with 1 μl of the diluted T4 endoV and incubated at 37 °C for 30 min. Reactions were terminated by adding one-fifth final volume of a solution containing 50% glycerol, 0.04% bromphenol blue, and 10% SDS. Subsequent to electrophoresis, the dried gels were analyzed as above. DNA replication products were purified from 30-min reactions and digested with the indicated restriction enzyme(s). Electrophoresis was first carried out at 0.4 V/cm for approximately 68 h in 0.4% agarose gels prepared in 45 mm Tris borate, 1 mm EDTA (TBE), also containing 0.2 μg/ml ethidium bromide. Excised lanes were cast into a second gel containing 1% agarose and 0.2 μg/ml ethidium bromide in TBE. The second electrophoresis, at a 90° angle with respect to the first, was run at 1.5 V/cm for approximately 24 h. The dried gels were exposed to a phosphor screen and scanned by a PhosphorImager™. AMBIS QuantProbe Software or ImageQuaNT™ (Molecular Dynamics) was used to determine the amount of radioactivity associated with DNA by volume integration of defined regions of the scanned images. Objects were drawn with the rectangle or polygon tool to closely surround the areas of interest. In single dimensional gel electrophoresis analyses, all DNA species were included within a single object for the measurement of total DNA synthesis; specific forms (e.g. RFI and RFII) were quantified individually. Lane specific background was defined by a rectangle drawn under the pUC19 band. Integrated volumes (net counts) were then normalized to pUC19 recovered in each lane. In experiments in which we incubated DNA with T4 endoV prior to gel electrophoresis, we noticed that we systematically lost a small fraction of the radiolabel incorporated at the ends of the linearized pUC19 molecules. Since this internal standard was added to each sample at the end of the in vitro replication reaction, the control (no enzyme addition) and treated samples contained the same ratio of total replication products to pUC19. We calculated this ratio for control samples and estimated the volume of pUC19 from the volume of total replication products for lanes containing T4 endoV. Then, this estimated volume of the internal standard was used to normalize the relative units of precursor incorporation in RFI and RFII. Analyses of the two-dimensional gel electrophoresis images acquired with the PhosphorImager™ were done with the polygon tool and ImageQuaNT™. Only replication-intermediate arcs were quantified; volume integration was done with no background correction. The integrated volumes of bubble and fork arcs were expressed as percentages of the sum of all replicating structures detected in each image. The percentage distribution determined with the dimer-containing molecule was compared with that observed with the undamaged DNA. Fig. 1 shows the position of thecis,syn-cyclobutane thymine dimer relative to the SV40 origin of replication in the double-stranded and closed circular molecule (7.4 kb) that was used as the site-specifically damaged substrate in the in vitro replication assays described below. These assays are dependent on Tag-directed initiation of replication at the SV40 origin and semi-conservative DNA synthesis (55Li J.J. Kelly T.J. Mol. Cell. Biol. 1985; 5: 1238-1246Google Scholar) by two replication forks moving away from the origin in opposite directions (bi-directional replication). Note that in this construct, the replication fork moving from the origin toward the singleEcoRI recognition site encounters the pyrimidine dimer (T[c,s]T) on the template to the leading strand of nascent DNA, 385 bp from the center of the SV40 origin of replication (Fig. 1). Dimer-containing and undamaged plasmids were replicated in vitro with extracts from human cells. Radiolabeled products were analyzed for the degree of inhibition of RFI synthesis by the pyrimidine dimer and to determine whether the T[c,s]T was present in the newly synthesized, closed circular DNA molecules. In this experimental system, the generation of dimer-containing RFI DNA could proceed along two potentially distinct pathways (Fig. 2). If the dimer blocks one of the forks, at least momentarily (Fig. 2 A), the other replication fork displacing in the opposite direction could complete synthesis around the circular DNA. This would generate an RFI from the undamaged strand and potentially leave the dimer in a gapped molecule. Following segregation of the daughter molecules, it is conceivable that other activities, such as gap-filling repair, could be responsible for completing synthesis across the pyrimidine dimer to form the dimer-containing RFI. Fig. 2 B depicts the displacement of the right replication fork up to and beyond the pyrimidine dimer, thus catalyzing bypass replication, presumably by extension of the leading strand. As both pathways depicted in Fig. 2 could be operational duringin vitro replication, it would be premature to conclude how bypass replication has taken place only from the position and orientation of the dimer in relationship to the origin of replication. Therefore, it became imperative to distinguish which pathway was more likely to be followed during replication of damaged DNA moleculesin vitro, to investigate potential mechanisms of dimer bypass and later evaluate biological consequences of placing the lesion on the template to the leading or lagging strand of nascent DNA. Fig. 3 illustrates the fractionation of in vitro replication products from reactions in which dimer-containing and control DNA were incubated with extracts from HeLa cells or XPV (CTag) fibroblasts. Results with extracts from other human fibroblasts are shown in Fig. 4. The amounts of total DNA replication products and RFI were estimated from their radioactivity (AMBIS™ or PhosphorImage™ units) after normalization to the internal standard (pUC19). Total DNA synthesis using the HeLa extract was not inhibited by the dimer. DNA synthesis on the damaged molecule averaged 98 ± 27% (mean ± S.D., n= 20) that on the undamaged molecule in experiments in which the incubation time varied from 1 to 6 h. In contrast, total DNA synthesis on the damaged molecule using the CTag extract was 69 ± 23% control (n = 15, p < 0.01, CTagversus HeLa, Student's t test). Synthesis of RFI DNA from the dimer-containing substrate was severely reduced in the CTag extract (27 ± 9%, n = 15) in comparison to HeLa (72 ± 22%, n = 19), and this difference was highly significant (p < 0.005). Thus, by two different measures of DNA synthesis, extracts from XPV cells were impaired relative to HeLa in their capacity to replicate a circular DNA molecule that contained a single pyrimidine dimer. Experiments with extracts from SV40-transformed fibroblasts (IDH4, SV80, XPA) yielded results that were comparable to those described for HeLa extracts. In the experiments illustrated in Fig. 4, for example, RFI synthesis from the T[c,s]T-containing molecules represented 86% (IDH4), 78% (SV80), and 53% (XPA) that determined with the undamaged control.Figure 4Extracts from human fibroblasts support the replication of T[c,s]T-containing plasmids, regardless of their NER capability. Template molecules containing the T[c,s]T dimer (lanes 1, 2, 5, 6, 9, and 10) or not (control inlanes 3, 4, 7, 8, 11, and 12) were replicatedin vitro during 2-h reactions with extracts from fibroblasts that are endowed with wild-type activity for NER (IDH4, lanes 1–4; SV80, lanes 5–8) or are defective in this process (XPA, NER-mutant, lanes 9–12). DNA was purified and incubated in the presence (+) or absence (−) of T4 endoV, as described under "Methods."View Large Image Figure ViewerDownload (PPT) RFI that contains a CPD is nicked by T4 endoV and converted to RFII with reduced electrophoretic mobility. By measuring the fraction of newly synthesized RFI DNA that is resistant to nicking by T4 endoV, one can calculate the fraction that carries the dimer (fraction sensitive to nicking). We determined that 0.43 ± 0.03 (n = 5) corresponded to the fraction of RFI DNA, produced during in vitro reactions (1.5 to 6 h) with the HeLa extract, that was nicked by T4 endoV (Fig. 3 and Table I). Under identical conditions, the nicked fraction of RFI synthesized from undamaged DNA by the same extract was 0.09 ± 0.08 (n = 5). By correcting for this background we found that 34% of the RFI DNA synthesized from the T[c,s]T-containing template by HeLa carried the dimer (theoretical maximum of 50%). Similar calculations with the data depicted in Fig. 4 revealed the presence of the dimer in 45, 52, and 54% of the RFI molecules newly synthesized by IDH4, SV80, and XPA extracts, respectively (Table I). In contrast, the small amount of RFI synthesized by the XPV (CTag) extract from the damaged molecule (Fig.3) demonstrated the same sensitivity to nicking by T4 endoV as the RFI products from undamaged DNA (Table I). We interpreted these results as evidence that the CTag extract was unable to complete synthesis of the template strand containing the pyrimidine dimer. Similar results were also obtained with XP30RO extracts (not shown).Table IT4 endonuclease V-sensitive sites in RFI DNA synthesized in vitroExtract sourceIncubation timeNicked fractionControl moleculeT[c,s]T-moleculeDimer-dependenthA. HeLa1.50.170.430.262.00.000.390.393.00.160.440.284.00.000.440.446.00.130.460.33Mean ± SD,0.09 ± 0.080.43 ± 0.030.34 ± 0.07B. IDH42.00.100.550.45C. SV802.00.000.520.52D. XPA2.00.000.540.54E. XPV (CTag)1.50.030.130.102.00.060.130.073.00.150.14−0.014.00.030.030.006.00.120.00−0.12Mean ± SD,0.08 ± 0.050.09 ± 0.070.01 ± 0.08DNA products of in vitro replication were incubated with T4 endoV to determine the fraction of RFI molecules containing the T[c,s]T (dimer-dependent nicked fraction). The amount of RFI remaining after treatment (normalized to the internal standard) was divided by the amount of RFI in identical samples incubated in the absence of the enzyme. This ratio (T4 endonuclease V-resistant fraction) was subtracted from 1 to determine the nicked fraction. The dimer-dependent fraction was the difference b
Referência(s)