C-to-G editing generates double-strand breaks causing deletion, transversion and translocation
Min HuangYining QinYafang ShangQian HaoChuanzong ZhanChaoyang LianSimin LuoLiu Daisy LiuSenxin ZhangYu ZhangYang WoNiu LiShuheng WuTuantuan GuiBinbin WangYifeng LuoYanni CaiXiaojing LiuZiye XuPengfei DaiSimiao Li‐SauerwineLiang ZhangJunchao DongJian WangXiaoqi ZhengYingjie XuYihua SunWei WuLeng-Siew YeapFei‐Long Meng
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Nuclear lamins are type V intermediate filament proteins. Lamins, including LA, LB1, LB2, and LC, are the major protein components forming the nuclear lamina to support the mechanical stability of the mammalian cell nucleus. Increasing evidence has shown that LA participates in homologous recombination (HR) repair of DNA double-strand breaks (DSBs) . However, the mechanisms underlying this process are incompletely understood. We recently identified the first lamin-binding ligand 1 (LBL1) that directly binds LA and inhibited cancer cell growth. We provided here further mechanistic investigations of LBL1 and revealed that LA interacts with the HR recombinase Rad51 to protect Rad51 from degradation. LBL1 inhibits LA–Rad51 interaction leading to accelerated proteasome-mediated degradation of Rad51, culminating in inhibition of HR repair of DSBs. These results uncover a novel post-translational regulation of Rad51 by LA and suggest that targeting the LA–Rad51 axis may represent a promising strategy to develop cancer therapeutics.
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DNA double-strand breaks (DSBs) are one of the most lethal types of DNA damage due to the fact that unrepaired or mis-repaired DSBs lead to genomic instability or chromosomal aberrations, thereby causing cell death or tumorigenesis. The classical non-homologous end-joining pathway (c-NHEJ) is the major repair mechanism for rejoining DSBs, and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a critical factor in this pathway; however, regulation of DNA-PKcs expression remains unknown. In this study, we demonstrate that miR-145 directly suppresses DNA-PKcs by binding to the 3′-UTR and inhibiting translation, thereby causing an accumulation of DNA damage, impairing c-NHEJ, and rendering cells hypersensitive to ionizing radiation (IR). Of note, miR-145-mediated suppression of DNA damage repair and enhanced IR sensitivity were both reversed by either inhibiting miR-145 or overexpressing DNA-PKcs. In addition, we show that the levels of Akt1 phosphorylation in cancer cells are correlated with miR-145 suppression and DNA-PKcs upregulation. Furthermore, the overexpression of miR-145 in Akt1-suppressed cells inhibited c-NHEJ by downregulating DNA-PKcs. These results reveal a novel miRNA-mediated regulation of DNA repair and identify miR-145 as an important regulator of c-NHEJ.
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Among various DNA damages, double‐strand breaks (DSBs) are considered as most deleterious, as they may lead to chromosomal rearrangements and cancer when unrepaired. Nonhomologous DNA end joining (NHEJ) is one of the major DSB repair pathways in higher organisms. A large number of studies on NHEJ are based on in vitro systems using cell‐free extracts. In this paper, we summarize the studies on NHEJ performed by various groups in different cell‐free repair systems.
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DNA double-strand breaks occur frequently in cycling cells, and are also induced by exogenous sources, including ionizing radiation. Cells have developed integrated double-strand break response pathways to cope with these lesions, including pathways that initiate DNA repair (either via homologous recombination or nonhomologous end joining), the cell-cycle checkpoints (G1-S, intra-S phase, and G2-M) that provide time for repair, and apoptosis. However, before any of these pathways can be activated, the damage must first be recognized. In this review, we will discuss how the response of mammalian cells to DNA double-strand breaks is regulated, beginning with the activation of ATM, the pinnacle kinase of the double-strand break signalling cascade.
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xthA- Escherichia coli, which are missing a major cellular apurinic/apyrimidinic (AP) endonuclease, are 5- to 10-fold more sensitive than xthA+ bacteria to mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) under conditions that induce the "adaptive response." The xthA(-)-dependent mutations are also dependent on SOS mutagenic processing and consist of both transversion and transition base substitutions. When MNNG-adapted xthA- bacteria are challenged with a high dose of MNNG, more xthA(-)-dependent SOS-dependent mutations are induced, and transversions are enhanced relative to transitions. The mutations induced by challenge are eliminated in xthA- alkA- bacteria, which are also deficient for 3-methyladenine glycosylase II activity. These data are consistent with the hypothesis that AP sites, at least some of which are produced by glycosylase activity, are mutagenic intermediates following cellular DNA alkylation.
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In mammalian cells, nonhomologous DNA end joining (NHEJ) is considered the major pathway of double-strand break (DSB) repair. Rejoining of DSB produced by decay of 125I positioned against a specific target site in plasmid DNA via a triplex-forming oligonucleotide (TFO) was investigated in cell-free extracts from Chinese hamster ovary cells. The efficiency and quality of NHEJ of the "complex" DSB induced by the 125I-TFO was compared with that of "simple" DSB induced by restriction enzymes. We demonstrate that the extracts are indeed able to rejoin 125I-TFO-induced DSB, although at approximately 10-fold decreased efficiency compared with restriction enzyme-induced DSB. The resulting spectrum of junctions is highly heterogeneous exhibiting deletions (1–30 bp), base pair substitutions, and insertions and reflects the heterogeneity of DSB induced by the125I-TFO within its target site. We show that NHEJ of125I-TFO-induced DSB is not a random process that solely depends on the position of the DSB but is driven by the availability of microhomology patches in the target sequence. The similarity of the junctions obtained with the ones found in vivo after125I-TFO-mediated radiodamage indicates that our in vitro system may be a useful tool to elucidate the mechanisms of ionizing radiation-induced mutagenesis and repair. In mammalian cells, nonhomologous DNA end joining (NHEJ) is considered the major pathway of double-strand break (DSB) repair. Rejoining of DSB produced by decay of 125I positioned against a specific target site in plasmid DNA via a triplex-forming oligonucleotide (TFO) was investigated in cell-free extracts from Chinese hamster ovary cells. The efficiency and quality of NHEJ of the "complex" DSB induced by the 125I-TFO was compared with that of "simple" DSB induced by restriction enzymes. We demonstrate that the extracts are indeed able to rejoin 125I-TFO-induced DSB, although at approximately 10-fold decreased efficiency compared with restriction enzyme-induced DSB. The resulting spectrum of junctions is highly heterogeneous exhibiting deletions (1–30 bp), base pair substitutions, and insertions and reflects the heterogeneity of DSB induced by the125I-TFO within its target site. We show that NHEJ of125I-TFO-induced DSB is not a random process that solely depends on the position of the DSB but is driven by the availability of microhomology patches in the target sequence. The similarity of the junctions obtained with the ones found in vivo after125I-TFO-mediated radiodamage indicates that our in vitro system may be a useful tool to elucidate the mechanisms of ionizing radiation-induced mutagenesis and repair. Mammalian genomes constantly suffer a variety of types of damage, of which double-strand breaks (DSB) 1The abbreviations used are: DSBdouble-strand break(s)bl.bluntcoh. cohesiveccc, covalently closed circleNHEJnonhomologous DNA end joiningocopen circlePupurinePypyrimidineRErestriction enzymeSSBsingle-strand break(s)TFOtriplex-forming oligoCHOChinese hamster ovaryMOPSO3-(N-morpholino)-2-hydroxypropanesulfonic acid 1The abbreviations used are: DSBdouble-strand break(s)bl.bluntcoh. cohesiveccc, covalently closed circleNHEJnonhomologous DNA end joiningocopen circlePupurinePypyrimidineRErestriction enzymeSSBsingle-strand break(s)TFOtriplex-forming oligoCHOChinese hamster ovaryMOPSO3-(N-morpholino)-2-hydroxypropanesulfonic acid are considered the most dangerous. DSB may arise spontaneously in the cell or may be induced by exogenous agents, such as ionizing radiation. The estimation that mammalian cells suffer at least 10 spontaneous DSB/day suggests that efficient repair of DSB is critical for cell survival (1.Haber J.E. Trends Genet. 2000; 16: 259-264Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar). Failure to do so can result in deleterious genomic rearrangements, cell cycle arrest, or cell death. double-strand break(s) blunt ccc, covalently closed circle nonhomologous DNA end joining open circle purine pyrimidine restriction enzyme single-strand break(s) triplex-forming oligo Chinese hamster ovary 3-(N-morpholino)-2-hydroxypropanesulfonic acid double-strand break(s) blunt ccc, covalently closed circle nonhomologous DNA end joining open circle purine pyrimidine restriction enzyme single-strand break(s) triplex-forming oligo Chinese hamster ovary 3-(N-morpholino)-2-hydroxypropanesulfonic acid Recent studies have revealed that DSB in the genomes of higher eukaryotes can be repaired by at least three different pathways (2.Pfeiffer P. Goedecke W. Obe G. Mutagenesis. 2000; 15: 289-302Crossref PubMed Scopus (338) Google Scholar): (i) Homologous recombination repair, the most accurate process, is able to restore the original sequence at the break. Because of its strict dependence on extensive sequence homology, this mechanism is suggested to be active mainly during the S and G2 phases of the cell cycle (3.Haber J.E. Trends Biochem. Sci. 1999; 24: 271-275Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 4.Haber J.E. Curr. Opin. Cell Biol. 2000; 12: 286-292Crossref PubMed Scopus (60) Google Scholar). (ii) Single-stranded annealing is another homology-dependent but less accurate process that can repair DSB between direct repeats and thereby produces mainly interstitial deletions (4.Haber J.E. Curr. Opin. Cell Biol. 2000; 12: 286-292Crossref PubMed Scopus (60) Google Scholar). (iii) Nonhomologous DNA end joining (NHEJ) comprises at least two different processes (5.Feldmann E. Schmiemann V. Goedecke W. Reichenberger S. Pfeiffer P. Nucleic Acids Res. 2000; 28: 2585-2596Crossref PubMed Scopus (188) Google Scholar). The major and best investigated NHEJ pathway depends on the Ku70/80 heterodimer, the catalytic subunit of the DNA-dependent protein kinase, DNA ligase IV, and its essential co-factor XRCC4 (6.Critchlow S.E. Jackson S.P. Trends Biochem. Sci. 1998; 23: 394-398Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar, 7.Featherstone C. Jackson S.P. Curr. Biol. 1999; 9: R759-R761Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). In contrast to homologous recombination repair and single-stranded annealing, NHEJ can operate in the absence of sequence homology (although short sequence homologies, so-called microhomologies, may facilitate the process) and is able to rejoin broken ends directly (2.Pfeiffer P. Goedecke W. Obe G. Mutagenesis. 2000; 15: 289-302Crossref PubMed Scopus (338) Google Scholar). This process is supposed to occur mainly in the G0 and G1 phases of the cell cycle and is considered to be the major pathway of DSB repair in mammalian cells, although it is typically accompanied by loss or gain of a few nucleotides. The regulation of these different pathways and their relative contributions to mammalian DSB repair have yet to be comprehended (1.Haber J.E. Trends Genet. 2000; 16: 259-264Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar). To elucidate the mechanisms of NHEJ, many studies have made use of restriction endonucleases (RE) to introduce defined DSB in the genomic DNA of cultured mammalian cells (8.Bryant P.E. Int. J. Radiat. Biol. Relat Stud. Phys. Chem. Med. 1985; 48: 55-60Crossref PubMed Scopus (134) Google Scholar, 9.Bryant P.E. Int. J. Radiat. Biol. Relat Stud. Phys. Chem. Med. 1984; 46: 57-65Crossref PubMed Scopus (271) Google Scholar, 10.Natarajan A.T. Obe G. Chromosoma. 1984; 90: 120-127Crossref PubMed Scopus (236) Google Scholar, 11.Obe G. Palitti F. Tanzarella C. Degrassi F. De Salvia R. Mutat. Res. 1985; 150: 359-368Crossref PubMed Scopus (45) Google Scholar, 12.Rouet P. Smih F. Jasin M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6064-6068Crossref PubMed Scopus (447) Google Scholar, 13.Rouet P. Smih F. Jasin M. Mol. Cell. Biol. 1994; 14: 8096-8106Crossref PubMed Scopus (591) Google Scholar) or in plasmids to be offered as DSB substrates in transfection assays (14.Roth D.B. Wilson J.H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3355-3359Crossref PubMed Scopus (109) Google Scholar, 15.Lutze L.H. Cleaver J.E. Morgan W.F. Winegar R.A. Mutat. Res. 1993; 299: 225-232Crossref PubMed Scopus (32) Google Scholar, 16.King J.S. Valcarcel E.R. Rufer J.T. Phillips J.W. Morgan W.F. Nucleic Acids Res. 1993; 21: 1055-1059Crossref PubMed Scopus (51) Google Scholar) or cell-free extracts (17.Daza P. Reichenberger S. Göttlich B. Hagmann M. Feldmann E. Pfeiffer P. Biol. Chem. Hoppe-Seyler. 1996; 377: 775-786Crossref PubMed Scopus (52) Google Scholar, 18.Pfeiffer P. Vielmetter W. Nucleic Acids Res. 1988; 16: 907-924Crossref PubMed Scopus (125) Google Scholar, 19.North P. Ganesh A. Thacker J. Nucleic Acids Res. 1990; 18: 6205-6210Crossref PubMed Scopus (119) Google Scholar, 20.Nicolas A.L. Munz P.L. Young C.S. Nucleic Acids Res. 1995; 23: 1036-1043Crossref PubMed Scopus (60) Google Scholar, 21.Baumann P. West S.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14066-14070Crossref PubMed Scopus (271) Google Scholar, 22.Labhart P. Eur. J. Biochem. 1999; 265: 849-861Crossref PubMed Scopus (60) Google Scholar). The fact that RE-induced DSB are exactly defined with respect to their structure (depending on the enzyme used: 5′- or 3′-overhangs or blunt ends; always 3′-hydroxyl and 5′-phosphate) and position within a given DNA sequence has greatly facilitated study of the efficiency and fidelity of DSB repair mechanisms in the above-mentioned systems by comparing the original DSB termini and the resulting repair site (junction). As opposed to such "clean" DSB, which are repaired very efficiently because they are accepted substrates of DNA-modifying enzymes, DSB generated by ionizing radiation or certain chemical agents are more complex and may, for instance, contain damaged sugar and base moieties and 5′-hydroxyl and 3′-phosphate groups. In addition, the investigation of the repair of such complex DSB on the molecular level is aggravated by the fact that these "dirty" DSB are usually randomly distributed and not positioned within a specific DNA sequence. Experimental approaches comprise the analysis of the mutational spectra generated by ionizing radiation or chemicals in selectable cellular genes (23.Phillips J.W. Morgan W.F. Mol. Cell. Biol. 1994; 14: 5794-5803Crossref PubMed Scopus (108) Google Scholar) and the use of oligonucleotides with unusual terminal structures in cell-free extracts (24.Beyert N. Reichenberger S. Peters M. Hartung M. Gottlich B. Goedecke W. Vielmetter W. Pfeiffer P. Nucleic Acids Res. 1994; 22: 1643-1650Crossref PubMed Scopus (25) Google Scholar) and plasmids carrying at their ends oligonucleotides damaged by bleomycin (25.Chen S. Inamdar K.V. Pfeiffer P. Feldmann E. Hannah M.F. Yu Y. Lee J.W. Zhou T. Lees-Miller S.P. Povirk L.F. J. Biol. Chem. 2001; 276: 24323-24330Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 26.Bennett R.A. Gu X.Y. Povirk L.F. Int. J. Radiat. Biol. 1996; 70: 623-636Crossref PubMed Scopus (35) Google Scholar, 27.Gu X.Y. Bennett R.A. Povirk L.F. J. Biol. Chem. 1996; 271: 19660-19663Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). A novel approach called gene-targeted radiotherapy has recently opened the possibility to target the radiodamage produced by Auger electron emitters such as 125I to a specific DNA sequence (as opposed to random targeting of total genomic DNA in traditional radiotherapy) (28.Panyutin I.G. Winters T.A. Feinendegen L.E. Neumann R.D. Q. J. Nucl. Med. 2000; 44: 256-267PubMed Google Scholar). Auger electron emitters are a large group of radioisotopes that decay by electron capture and/or conversion emitting a cascade of low energy electrons that produces a highly charged daughter atom. The combined effect of low energy electrons and positively charged daughter atoms results in highly localized damage to the molecular structures within a short range from the decay site (Auger effect). Decay of 125I results in emission of, on average, 21 electrons and produces a correspondingly positively charged tellurium atom. Incorporated into DNA, the decay of125I produces DSB localized mostly within one turn of the double-helix around the decay site (10 bp) with an efficiency of 0.8 DSB/decay. This extremely short range of radiodamage produced by125I led to the idea of targeting this Auger electron emitter to specific genes within genomic or plasmid DNA (29.Sedelnikova O.A. Luu A.N. Karamychev V.N. Panyutin I.G. Neumann R.D. Int. J. Radiat. Oncol. Biol. Phys. 2001; 49: 391-396Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Sequence-specific delivery of 125I-induced radiodamage is achieved by the use of triplex-forming oligonucleotides (TFO), short single-stranded oligonucleotides capable of forming triple helixes (triplexes) with polypurine:polypyrimidine sequences. In such triplexes, the TFO occupies the major groove of the target double-helix and forms Hoogsteen hydrogen bonds with the purines of the Watson-Crick base pairs. The specificity of sequence recognition is comparable with that provided by complementary Watson-Crick base pairing (30.Panyutin I.G. Neumann R.D. Nucleic Acids Res. 1997; 25: 883-887Crossref PubMed Scopus (52) Google Scholar, 31.Panyutin I.G. Neumann R.D. Acta Oncol. 1996; 35: 817-823Crossref PubMed Scopus (49) Google Scholar, 32.Panyutin I.G. Neumann R.D. Nucleic Acids Res. 1994; 22: 4979-4982Crossref PubMed Scopus (81) Google Scholar). To investigate the repair of site-specific 125I-induced DSB, a TFO labeled on its 3′-end with 125I (125I-TFO) was used to introduce DSB within its target sequence on plasmid pUC19-MDR1 (33.Panyutin I.V. Luu A.N. Panyutin I.G. Neumann R.D. Radiat. Res. 2001; 156: 158-166Crossref PubMed Scopus (24) Google Scholar). The linearized plasmid was incubated with cell-free extracts from CHO cells capable of performing efficient NHEJ (5.Feldmann E. Schmiemann V. Goedecke W. Reichenberger S. Pfeiffer P. Nucleic Acids Res. 2000; 28: 2585-2596Crossref PubMed Scopus (188) Google Scholar). We show that the repair of the125I-induced DSB is about a factor of 10 less efficient than the repair of RE-induced DSB. The resulting spectrum of junctions shows deletions of varying sizes resembling the ones found in selectable genes after irradiation of mammalian cells with ionizing radiation. Our study may contribute to the understanding of how the damage produced by Auger electron emitters is repaired by mechanisms of NHEJ, which is important for their application in gene-targeted radiotherapy. The two wild-type Chinese hamster ovary cell lines, CHO-K1 and AA8, were grown at 37 °C in a humidified 5% CO2atmosphere in Ham's F-12 medium enriched with 10% fetal calf serum, 2 mml-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. Whole cell extracts from CHO-K1 and AA8 cells were prepared exactly as described previously (5.Feldmann E. Schmiemann V. Goedecke W. Reichenberger S. Pfeiffer P. Nucleic Acids Res. 2000; 28: 2585-2596Crossref PubMed Scopus (188) Google Scholar, 17.Daza P. Reichenberger S. Göttlich B. Hagmann M. Feldmann E. Pfeiffer P. Biol. Chem. Hoppe-Seyler. 1996; 377: 775-786Crossref PubMed Scopus (52) Google Scholar). In each preparation, ∼5 × 108 cells of each cell line were used to yield 0.5–1 ml of extract with a protein concentration ranging between 6–10 mg/ml. The extracts were stored in 50-μl aliquots in liquid nitrogen and remained active for 6–12 months. Directly prior to use in the NHEJ reaction, the extract aliquot was dialyzed against freshly prepared M buffer (50 mm MOPSO-NaOH, pH 7.5, 40 mmKCl, 10 mm MgCl2, 5 mm2-mercaptoethanol) on microdialysis filters (0.025-μm pore diameter; catalog number VSWPO2500; Millipore) for 30 min at 4 °C. Labeling of the TFO with125I-dC was performed by extension of the 3′-end of a primer in the presence of 125I-dCTP (PerkinElmer Life Sciences) and Klenow fragment of DNA polymerase I as described previously (33.Panyutin I.V. Luu A.N. Panyutin I.G. Neumann R.D. Radiat. Res. 2001; 156: 158-166Crossref PubMed Scopus (24) Google Scholar). To form a triplex, topoisomerase-relaxed pUC19-MDR1, a 2727-bp derivative of pUC19 containing a 32-bp polypurine-polypyrimidine fragment from the MDR1 gene as TFO-target sequence (see Fig. 1 and Refs. 33.Panyutin I.V. Luu A.N. Panyutin I.G. Neumann R.D. Radiat. Res. 2001; 156: 158-166Crossref PubMed Scopus (24) Google Scholar and 34.Sedelnikova O.A. Panyutin I.G. Luu A.N. Reed M.W. Licht T. Gottesman M.M. Neumann R.D. Antisense Nucleic Acid Drug Dev. 2000; 10: 443-452Crossref PubMed Scopus (26) Google Scholar) was mixed with purified125I-TFO in 30 mm NaAc buffer, pH 5.0, and heated to 70 °C for 3 min followed by slow cooling to room temperature. For the accumulation of 125I decays, the sample was stored at −70 °C. After a period of 60 days (the half-life of 125I), about 50% of total covalently closed circular (ccc) pUC19-MDR1 was converted to open circle (oc), and about 20% was converted to linear DNA indicative of double-strand breakage of the plasmid as estimated by separation of the products in 1.5% agarose gels containing ethidium bromide. To remove contaminating oc and ccc DNA, the linear form of pUC19-MDR1 was purified twice over 1.5% preparative low melting point NuSieve agarose (BioProducts FMC) gels in TAE buffer (40 mm Tris-HAc, pH 7.4, 12 mm NaAc, 0.1 mm EDTA) containing 0.5 μg/ml ethidium bromide. Electrophoresis was performed at 2 V/cm for 24 h with continuously recirculated TAE buffer containing 0.5 μg/ml ethidium bromide, and separation of DNA in oc, linear, and ccc forms was visualized under UV light. Linear DNA was purified using the Agar ACETM agarose-digesting enzyme (Promega) according to the manufacturer's instructions. The samples were purified further by two extractions with phenol and phenol:chloroform:isoamyl alcohol (25:24:1; Invitrogen) and precipitated with ethanol. After resuspension in 50 μl of TE (10 mm Tris-HCl, pH 7.6, 0.1 mmEDTA) the samples were finally purified by gel filtration through G-50 Microspin columns (Amersham Biosciences). The resulting linearized pUC19-MDR1 substrate used in extract joining assays was found to contain on the average less than 5% of contaminating oc DNA and no ccc DNA at all. The three substrates for cohesive (coh.) and blunt (bl.) end ligation were derived from pUC19-MDR1 by digestion with a single restriction enzyme (BamHI: 5′-coh.;PstI: 3′-coh.; HincII: bl.). The five substrates containing noncomplementary ends were derived from a 4-kb modified pUC19-MDR1-λ construct harboring a 1.25-kb fragment of λ-DNA between the restriction sites used for substrate preparation. Generation of substrates containing two noncomplementary ends was controlled by quantitative excision of the λ insert. Each substrate was named after the pair of RE used in its preparation (Eco/Asp, 5′/5′; Sac/Kpn, 3′/3′; Eco/Sma, 5′/bl.;Sac/Sma, 3′/bl.; Eco/Kpn, 5′/3′). All RE-linearized substrates were gel-purified using a gel extraction kit (Qiagen). In standard reactions, 10 ng of 125I-TFO- or RE-linearized plasmid substrate, respectively, were incubated for up to 360 min at 25 °C in a total volume of 10 μl containing 6–8 μg/μl of extract protein in M buffer supplemented with 1 mm ATP, pH 7.5, and 200 μm dNTPs (50 μm each) and 50 ng/μl bovine serum albumin. The reactions were terminated by adjustment to 20 mm Tris-HCl, pH 7.5, 10 mm EDTA, 1% SDS and incubation at 65 °C for 5 min. After digestion for 30 min at 37 °C with 2 mg/ml proteinase K, equivalents of 2 ng of substrate DNA were electrophoresed in 1% agarose gels in the presence of 1 μg/ml ethidium bromide to separate oc from ccc products and visualized by in situ gel hybridization (35.Thode S. Schäfer A. Pfeiffer P. Vielmetter W. Cell. 1990; 60: 921-928Abstract Full Text PDF PubMed Scopus (174) Google Scholar) using a pUC19-specific probe labeled with [32P]α-dCTP by random priming. Reaction products were quantified in a phosphorimaging facility (Packard Bioscience) as percentages of the total radioactivity/lane. Circular joined products were cloned by transformation of 4-ng equivalents of substrate DNA of each NHEJ sample in Escherichia coli strain DH5α to yield single clones that were purified by miniscale extraction. In the case of 125I-TFO-linearized pUC19-MDR1, the samples were digested with BglII prior to transformation to remove oc contaminants originating from substrate preparation that could yield false positives. Clones from 125I-TFO-linearized pUC19-MDR1 were subjected again to cleavage with BglII, and only BglII-resistant clones were analyzed by sequencing (Seqlab). The clones from ligation products (Bam, Pst, and Sma) were subjected to cleavage with the original RE to check for accurate ligation. The clones from NHEJ products (Eco/Asp, Sac/Kpn, Eco/Sma, Sac/Sma, and Eco/Kpn) were analyzed directly by sequencing (ABI Prism 377 DNA Sequencer; PerkinElmer Life Sciences). For the analysis of dimer products from 125I-TFO-linearized pUC19-MDR1, the dimer band was gel-purified using a gel extraction kit (Qiagen). Dimer junctions were amplified by PCR with 2.5 units of Taq polymerase in Taq buffer (MBI Fermentas) in a total volume of 50 μl containing 1 ng of dimer product, 20 pmol of each primer (pUC19-MDR1-For, 5′-GGGGCCTCTTCGCTATTACG; pUC19-MDR1-Rev, 5′-AGGCACCCCAGGCTTTACACTTTA), 2.5 mm MgCl2, and 200 μm of each dNTP. PCR was performed in a thermocycler (PerkinElmer Life Sciences) for 30 cycles (30 s 95 °C; 30 s 54 °C; 1 min 72 °C). The resulting 300-bp PCR product was digested with BglII to remove PCR products possibly originating from oc contaminants. BglII-resistant PCR product was gel-purified and subcloned using a cloning kit (Invitrogen). The resulting clones were purified by miniscale extraction and subjected again to cleavage with BglII, and only BglII-resistant clones were analyzed by sequencing. For the diagrams in Fig. 8 (A and B), the following calculations were performed. The distribution of breaks around the125I decay site had been measured previously as single-strand breaks (SSB) occurring in the Pu-rich and Py-rich strand, respectively (33.Panyutin I.V. Luu A.N. Panyutin I.G. Neumann R.D. Radiat. Res. 2001; 156: 158-166Crossref PubMed Scopus (24) Google Scholar) (see bars in Fig. 1) and is given here as the average probability ((Pu + Py)/2) of all types of DSB (gray bars in Fig. 8, A and B; see "Discussion" for details) to occur at a given base pair position. The relative frequencies for the occurrence of the breakpoints of junctions 2–34 (see Fig. 5) at a particular nucleotide (black bars in Fig. 8A) were calculated as follows: (i) Blunt junctions. The number of a particular junction was normalized to the total number of junctions (64) and divided by two because the breakpoint can be either counted to the left or to the right side of the deletion (e.g. junction 8 in Fig. 5); because this junction occurred twice, its relative frequency would be 2/64 = 0.0313. Because the breakpoint can be counted either to the A on the left side or to the C on the right side, the relative frequency of this breakpoint at the A and C, respectively, is 0.0156. (ii) Microhomology junctions. The calculation was performed as for blunt junctions with the additional inclusion of a factor for the microhomology (e.g. junction 23 in Fig. 5) that occurs twice and exhibits a 2-bp homology (AG) with three possible breakpoints. Therefore, the relative frequency of the breakpoints would be 2/64 × 2 × 3= 0.0052 for any nucleotide within the microhomology and each of the nucleotides flanking the microhomology on the left and right side, respectively (A, A, and G on the left side and the A, G, and T, on the right side). Each black bar in the diagram represents the sum of the relative frequencies of all breakpoints occurring at a particular nucleotide of the target sequence. The χ2 test was performed for Fig. 8A. Multiplication of the average probability of a DSB at a given base pair by the number of total junctions [(Pu + Py)/2] × 4 yields the expected frequency (E) of a junction to occur at this base pair, which was compared with the observed frequency (O) of junctions occurring at this position. The χ2 value was calculated using the formula (O −E)2/E. Because the distribution of observed junctions spans a larger sequence region (27 bp) than the distribution of DSB (19 bp; 5′-GAAG…… GAGT), only the junctions falling into this 19-bp region were taken into account resulting in 19 categories yielding a degree of freedom of 18. The estimated χ2 value is 49.54 (Σ(O −E)2/E) and significantly larger than 28.87, the value for the 5% interval for the degree of freedom of 18. Therefore, the hypothesis that junction formation is a random process that follows the distribution of the 125I-TFO-induced DSB has to be rejected (see "Discussion"). The distribution of nucleotides deleted around the decay site (see black bars in Fig. 8B) was calculated as follows. In the 64 junctions (see Fig. 4, junctions 2–34), a total of 422 nucleotides were deleted (e.g. the G* in the target sequence that was lost unambiguously in 41 cases and was part of a microhomology in 14 cases; see the dots in the sequences of Fig. 5). Because it was unknown from which of the two DSB ends the G in the corresponding microhomology originated, 14 was divided by two (14/2 = 7) so that the relative frequency at which the G* is lost in all 64 junctions is (41+7)/422 = 0.1137. Each black bar in the diagram represents the relative frequency with which a particular nucleotide was deleted from the target sequence. Annealing the 125I-TFO to its target sequence within pUC19-MDR1 and subsequent incubation for 60 days at −70 °C yield sequence-specific DSB within a short region of about 10 bp in each direction opposite the 125I-dC within the unique BglII site (A/GATCT) of the plasmid (Fig. 1). The distribution and relative frequencies of breaks had been determined previously by analysis of the SSB occurring in the Pu- and Py-rich strand, respectively, which is indicated schematically in Fig. 1 (33.Panyutin I.V. Luu A.N. Panyutin I.G. Neumann R.D. Radiat. Res. 2001; 156: 158-166Crossref PubMed Scopus (24) Google Scholar). The slightly asymmetric distribution of SSB in the two strands reflects the structure of the Py motif triple helix. Gel-purified 125I-TFO-linearized pUC19-MDR was subjected to DNA end joining in cell-free extracts from CHO-K1 and AA8 cells as described under "Experimental Procedures." For comparison, extract joining reactions were also carried out with pUC19-MDR1 linearized by restriction endonucleases. NHEJ reaction products were separated in agarose gels, and the corresponding repair sites (junctions) were cloned in E. coli for subsequent sequence analysis. To determine the efficiency of NHEJ of the 125I-TFO-linearized substrate, we used different RE-linearized substrates for comparison. Substrates generated by cleavage with a single RE have compatible ends that allow measurement of the efficiency of ligation of cohesive 5′- (Bam) or 3′-ends (Pst), respectively, or blunt ends (HincII). Substrates generated by cleavage with two different RE have noncomplementary DNA ends (Eco/Asp, 5′/5′; Sac/Kpn, 3′/3′; Eco/Sma, 5′/bl.;Sac/Sma, 3′/bl.; Eco/Kpn, 5′/3′) that allow measurement of the efficiency of genuine nonhomologous end joining. This type of end joining is more complex and requires more factors than "simple" cohesive or blunt end ligation because the ends must be converted first into a ligatable form by DNA fill-in synthesis and/or exonucleolytic removal of nonmatching bases (36.Pfeiffer P. Toxicol. Lett. 1998; 96–97: 119-129Crossref PubMed Scopus (41) Google Scholar) (Fig. 2 and below). Rejoining of125I-TFO-induced DSB is expected to be even more complex because these dirty breaks may contain damaged sugar and base moieties, 5′-hydroxyl and 3′-phosphate groups that are not substrates for DNA-modifying enzymes such as DNA ligase or DNA polymerase and therefore must be removed prior to NHEJ (25.Chen S. Inamdar K.V. Pfeiffer P. Feldmann E. Hannah M.F. Yu Y. Lee J.W. Zhou T. Lees-Miller S.P. Povirk L.F. J. Biol. Chem. 2001; 276: 24323-24330Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 27.Gu X.Y. Bennett R.A. Povirk L.F. J. Biol. Chem. 1996; 271: 19660-19663Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). In addition to that, it is important to note that each RE substrate contains only a single type of DSB with ends exactly defined in structure and sequence. In contrast, the 125I-TFO substrate represents a mixture of molecules containing many different types of DSB because of the fact that the 125I-TFO induces multiple breaks distributed along a 19-bp region (see also "Discussion" and Fig. 8). Therefore, the term "complex DSB" used below not only includes the presumptive dirty DSB but also a large variety of DSB ends differing in structure and sequence. The extract-mediated NHEJ reaction converts all three different substrate types into monomeric oc reaction intermediates, ccc products, and linear multimers (mostly dimers), which are readily separated in agarose gels. In standard reactions, about 30–50% of the RE substrate input are converted into ccc and dimer products and the ratio of ccc:dimer product is ∼2:1 (but may vary with the batch of extract used and other factors like protein concentration and DNA concentration). We did not find any quantitative or qualitative differences between the CHO-K1 extract and the AA8 extract. A representative example of the reaction kinetics of three of the eight RE substrates and the 125I-TFO substrate is given in Fig. 3. As reflected by the levels of ccc product formation after 6 h at 25 °C, the reaction is most efficient with the ligation of cohesive (Pst) and blunt ends (HincII) that converts on the average 37% of the input substrate into ccc product (and 12% into dimers). Rejoining of noncomplementary RE ends (Eco/Kpn) is somewhat less efficient and converts on the average 29% of the linear inp
Non-homologous end joining
Sequence (biology)
Double strand
Homology directed repair
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