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End Modification of a Linear DNA Duplex Enhances NER-Mediated Excision of an Internal Pt(II)-Lesion Tracey McGregor Mason,† Michael B. Smeaton,† Joyce C. Y. Cheung, Les A. Hanakahi, and Paul S. Miller* Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, Maryland 21205. Received November 27, 2007; Revised Manuscript Received February 11, 2008
The study of DNA repair has been facilitated by the development of extract-based in Vitro assay systems and the use of synthetic DNA duplexes that contain site-specific lesions as repair substrates. Unfortunately, exposed DNA termini can be a liability when working in crude cell extracts because they are targets for DNA end-modifying enzymes and binding sites for proteins that recognize DNA termini. In particular, the double-strand break repair protein Ku is an abundant DNA end-binding protein that has been shown to interfere with nucleotide excision repair (NER) in Vitro. To facilitate the investigation of NER in whole-cell extracts, we explored ways of modifying the exposed ends of synthetic repair substrates to prevent Ku binding and improve in Vitro NER efficiency. Replacement of six contiguous phosphodiester linkages at the 3′-ends of the duplex repair substrate with nucleaseresistant nonionic methylphosphonate linkages resulted in a 280-fold decrease in binding affinity between Ku and the modified duplex. These results are consistent with the published crystal structure of a Ku/DNA complex [Walker et al. (2001) Nature 412, 607-614] and show that the 3′-terminal phosphodiester linkages of linear DNA duplexes are important determinants in DNA end-binding by Ku. Using HeLa whole-cell extracts and a 149-base pair DNA duplex repair substrate, we tested the effects of modification of exposed DNA termini on NER-mediated in Vitro excision of a 1,3-GTG-Pt(II) intrastrand cross-link. Methylphosphonate modification at the 3′-ends of the repair substrate resulted in a 1.6-fold increase in excision. Derivatization of the 5′-ends of the duplex with biotin and subsequent conjugation with streptavidin to block Ku binding resulted in a 2.3-fold increase excision. By combining these modifications, we were able to effectively reduce Ku-derived interference of NER excision in Vitro and observed a 4.4-fold increase in platinum lesion excision. These modifications are easy to incorporate into synthetic oligonucleotides and may find general utility whenever synthetic linear duplex DNAs are used as substrates to investigate DNA repair in whole-cell extracts.
INTRODUCTION Studies of DNA repair often use damaged plasmid DNA or lesion-containing, double-stranded, linear DNA duplexes as repair substrates. Plasmid DNA is problematic as a substrate for in Vitro repair studies because the size of the substrate makes it difficult to characterize and quantify the repair process. Unlike plasmid DNA, synthetic linear duplexes that contain site-specific lesions are readily synthesized and can be labeled in a variety of ways for high-sensitivity detection of repair activity. Such synthetic linear DNA duplexes provide convenient and easily manipulatable substrates for carrying out detailed studies of DNA repair. For example, linear DNA duplexes of ∼150 bp in length that contain a 1,3-diammineplatinum(II) intrastrand crosslink have been instrumental in the study of nucleotide excision repair (NER) in whole-cell extracts derived from Chinese hamster ovary (CHO) and human (HeLa) cell lines (1, 2). Similar synthetic DNA duplexes have been employed in the study of psoralen interstrand cross-link repair in whole-cell extracts and in fully reconstituted reactions using purified repair proteins (3–6). A potential problem with using linear duplexes in repair studies is the presence of exposed DNA termini, which are * Correspondence should be addressed to Paul S. Miller, Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, 615 North Wolfe Street, Baltimore, MD 21205. E-mail:
[email protected], Phone: (410)-955-3489, Fax: (410)-9552926. † These authors contributed equally to this work.
targets for DNA end-modifying enzymes, such as nucleases or phosphatases, and binding sites for proteins that recognize DNA ends. In particular, proteins involved in the recognition and repair of DNA double-strand breaks (DSB) have been shown to bind at exposed DNA termini of synthetic duplexes (7). The DSB repair protein Ku70/80 (Ku), which participates in both DSB repair and in immunoglobulin gene recombination, is a DNA end-binding protein that is abundant in whole-cell extracts (8–12). Previous studies have suggested that Ku not only binds DNA ends, but once bound can translocate along the DNA duplex. This makes it possible for several Ku molecules to occupy a single linear duplex and to potentially interfere with damage recognition or processing (13). In this article, we present strategies that can improve efficiency in extract-based DNA repair assay systems by preventing recognition of DNA termini by end-binding and -modifying factors. Introduction of nuclease-resistant, nonionic methylphosphonate linkages at the 3′-termini of the duplexes significantly reduces Ku binding. Derivatization of the 5′-ends of the duplex with biotin and subsequent conjugation with streptavidin is known to block binding of Ku to linear DNA duplexes (14). We have found that, in combination, these two modifications significantly enhance NER-mediated excision of an internal 1,3-Pt(II) intrastrand cross-link from a 149 bp linear duplex when incubated with a HeLa whole cell extract.
EXPERIMENTAL PROCEDURES Protected deoxyribonucleoside 3′-O-phosphoramidites, 5′biotin phosphoramidite, and oligonucleotide synthesis reagents
10.1021/bc7004363 CCC: $40.75 2008 American Chemical Society Published on Web 05/01/2008
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Table 1. Linear DNA Duplex Sequences
a Methylphosphonate internucleotide linkages are located on the 3′-side of the nucleosides shown in bold italics. G indicates a platinated G residue. The component duplexes that were used to form the full-length duplexes are separated by dashes.
were purchased from Glen Research, Inc. Protected deoxyribonucleoside-3′-O-methylphosphonamidites were a product of JBL, Inc. Cis-diamminedichloroplatinum (II) was purchased from Sigma-Aldrich, Inc. Polynucleotide kinase and T4 DNA ligase were obtained from New England Biolabs, Inc. Reactions employing these enzymes were carried out in buffer supplied by the manufacturer. High performance liquid chromatography (HPLC) was performed on a Varian instrument using a 0.4 × 25 cm Dionex strong anion exchange (SAX) column. MALDITOF mass spectra were obtained on an Applied Biosystems Voyager mass spectrometer at the AB Mass Spectrometry/ Proteomics Facility, Johns Hopkins School of Medicine, with support from a National Center for Research Resources shared instrumentation grant 1S10-RR14702. Talon metal affinity resin beads were purchased from BD Biosciences, Inc., and the Hitrap heparin and Hi-trap Q columns were obtained from General Electric Health Sciences, Inc. Coomassie Fluor Orange was a product of Molecular Probes, Inc. Surface plasmon resonance experiments were performed on a BIACORE 3000 instrument. Streptavidin-coated sensor chips (Sensor Chip SA) and Surfactant P20 were purchased from Biacore, Inc. Phosphorimage screens were read on a Typhoon 9200 phosphorimager. Oligonucleotide Synthesis. The sequences of the oligodeoxyribonucleotides used in these studies are shown in Table 1. The oligonucleotides and biotin-conjugated oligonucleotides were synthesized on controlled pore glass (CPG) supports by standard phosphoramidite chemistry using an Applied Biosystems model 392 or model 3400 DNA/RNA synthesizer. Phosphoramidite concentrations were 0.15 M; coupling times were 120 s; and the activator was 5-ethylthiotetrazole. In the case of the methylphosphonate-derivatized oligonucleotides, the standard oxidizing agent was replaced with a solution containing 1.27 g iodine, 12.5 mL 2,6-lutidine, and 100 µL water in 37.5 mL tetrahydrofuran, and standard Cap B reagent was replaced with a solution containing 1.25 g of 4-N,N′-dimethylaminopyridine in 50 mL of anhydrous pyridine. The DNA synthesizer was programmed to remove the 5′-terminal dimethoxytrityl
group after the final coupling step. The oligonucleotides were cleaved from their CPG supports and deprotected by treating the support with 400 µL of 95% ethanol/concentrated ammonium hydroxide (1:3 v/v) at 55 °C for 3.5 h. In the case of the methylphosphonate-derivatized oligonucleotides, a two-step deprotection procedure was used. The CPG support was first treated with 95% ethanol/concentrated ammonium hydroxide (1:3 v/v) for 2.5 h at room temperature. The supernatant was removed from the support and evaporated. The residue was treated with a solution containing 22.5 µL 95% ethanol, 22.5 µL acetonitrile, and 50 µL ethylenediamine for 6 h at room temperature. The solution was cooled to 4 °C and neutralized with 600 µL of ice-cold 2 N hydrochloric acid. The oligonucleotide was then desalted on a C-18 SEP PAK cartridge (Waters, Inc.) or C-18 Clarity column (Phenomenex, Inc.) and eluted from the column with 50% aqueous acetonitrile. Oligonucleotides 30 nucleotides in length or shorter were purified by SAX HPLC using a 30 min linear gradient of 0-0.5 M sodium chloride at a flow rate of 1 mL/min. Longer oligonucleotides were purified on 20 cm × 20 cm × 0.75 mm 20% polyacrylamide gels containing 7 M urea in a TBE running buffer that consisted of 89 mM Tris, 89 mM boric acid, and 2 mM ethylenediaminetetraacetic acid, pH 8.0. The oligonucleotides were located on the gel by UV shadowing and extracted from the gel by incubating the gel overnight at 37 °C in a buffer containing 20% acetonitrile in 0.1 M ammonium acetate, pH 6.2. The gel-purified oligonucleotides were desalted on C-18 cartridges as described above and their compositions were confirmed by MALDI-TOF mass spectrometry. Platinated oligonucleotide X (upper strand of duplex X) was prepared by incubating 26.5 nmol of the oligonucleotide in a solution containing 36 µM (54 nmol) cis-diamminedichloroplatinum (II) in 1.5 mL of Tris-EDTA buffer at 37 °C for 16 h. The reaction mixture was desalted using a SEP PAK cartridge and the platinated oligonucleotide, whose retention time is less than that of the nonplatinated oligomer, was purified by SAX HPLC using a linear gradient of 0.0-0.5 M sodium chloride in
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buffer containing 20% acetonitrile in 50 mM Tris, pH 7.8. The purified oligonucleotide was desalted on a SEP PAK cartridge and its composition was confirmed by MALDI-TOF mass spectrometry, m/z calcd 4078.64, m/z found 4079.36. Preparation of Oligonucleotide Duplexes. Duplexes 1-3 were prepared by annealing their component oligonucleotides in a buffer containing 70 mM Tris, pH 7.5, 10 mM magnesium chloride, and 5 mM dithiothreitol. The 149-mer duplexes 4-6 were prepared by ligation of their component duplexes following a procedure described by Huang et al. (1) The upper, platinated strand of duplex X (10 pmol) was first phosphorylated with [γ-32P] ATP (6000 Ci/mmol) using T4 polynucleotide kinase. After excess ATP was removed using a G-10 spin column, the phosphorylated duplex was annealed to 15 pmol of its phosphorylated complementary strand to form duplex X. This duplex was then mixed with 15 pmol each of the appropriate phosphorylated component duplexes in 1X ligase buffer (New England Biolabs, Inc.) supplemented with 1 mM ATP incubated with 1000 units of T4 DNA ligase for 30 min at room temperature followed by overnight incubation at 16 °C. The reaction mixture was concentrated to
