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repair of uniquely placed 1,2-GG, 1,2-AG, and 1,3-GTG cisplatin-crosslinks, and a 2-acetylaminofluorene lesion. The 1,3 crosslink and the acetylaminofluorene.
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Nucleic Acids Research, 1997, Vol. 25, No. 3

 1997 Oxford University Press

Differential human nucleotide excision repair of paired and mispaired cisplatin-DNA adducts Jonathan G. Moggs, David E. Szymkowski1, Masami Yamada2, Peter Karran and Richard D. Wood* Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, UK, 1Roche Research Centre, Welwyn Garden City, Hertfordshire AL7 3AY, UK and 2Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158, Japan Received November 4, 1996; Revised and Accepted December 11, 1996

ABSTRACT In order to understand the action of the chemotherapeutic drug cisplatin, it is necessary to determine why some types of cisplatin-DNA intrastrand crosslinks are repaired better than others. Using cell extracts and circular duplex DNA, we compared nucleotide excision repair of uniquely placed 1,2-GG, 1,2-AG, and 1,3-GTG cisplatin-crosslinks, and a 2-acetylaminofluorene lesion. The 1,3 crosslink and the acetylaminofluorene lesion were repaired by normal cell extracts ∼15–20 fold better than the 1,2 crosslinks. No evidence was found for selective shielding of 1,2 cisplatin crosslinks from repair by cellular proteins. Fractionation of cell extracts to remove putative shielding proteins did not improve repair of the 1,2-GG crosslink, and cell extracts did not selectively inhibit access of UvrABC incision nuclease to 1,2-GG crosslinks. The poorer repair of 1,2 crosslinks in comparison to the 1,3 crosslink is more likely a consequence of different structural alterations of the DNA helix. In support of this, a 1,2-GG-cisplatin crosslink was much better repaired when it was opposite one or two non-complementary thymines. Extracts from cells defective in the hMutSα mismatch binding activity also showed preferential repair of the 1,3 crosslink over the 1,2 crosslink, and increased repair of the 1,2 adduct when opposite thymines, showing that hMutSα is not involved in the differential NER of these substrates in vitro. Mismatched cisplatin adducts could arise by translesion DNA synthesis, and improved repair of such adducts could promote cisplatin-induced mutagenesis in some cases. INTRODUCTION Cisplatin is used with varying success for the treatment of human cancers. Although 90% of testicular cancers can be cured by cisplatin chemotherapy, a significant problem is the development of resistant tumours (1,2). The basis for the therapeutic effectiveness of cisplatin is not fully understood but its cytotoxic action against tumour cells is thought to be mediated through the

* To

whom correspondence should be addressed

formation of cisplatin-DNA adducts which may inhibit DNA replication and/or transcription (3). Cisplatin forms primarily 1,2-intrastrand crosslinks between adjacent purines in DNA, and also introduces other adducts including 1,3 crosslinks, interstrand crosslinks and monoadducts. The main mechanism for removing intrastrand crosslinks is nucleotide excision repair (NER), but the efficiency of removal varies among different intrastrand crosslinks both in vivo (4,5) and in vitro (6–9). NER in human cells involves recognition of damage by factors that include the XPA and RPA proteins, incision by the structure-specific endonucleases XPG on the 3′ side of a lesion and ERCC1/XPF on the 5′ side, and repair DNA synthesis mediated by a PCNA-dependent DNA polymerase (10,11). In order to better understand the toxic and mutagenic properties of cisplatin, it is important to investigate the reasons for the differential repair of intrastrand cisplatin-DNA crosslinks. Several explanations have been proposed. One hypothesisis is that the specific binding of cellular proteins, notably HMG-box proteins, to 1,2-intrastrand cisplatin-DNA crosslinks could shield these lesions from recognition by NER (3,12). Alternatively, 1,2 crosslinks might titrate essential DNA-binding proteins such as the transcription factor hUBF away from their natural sites of action (13). The simplest explanation, however, is that the extent of DNA structural alteration caused by a particular lesion dictates the efficiency of damage recognition (6,14). We report here that a more distorting 1,3-intrastrand d(GpTpG)-cisplatin crosslink is repaired much more readily by human whole cell extracts than a less distorting 1,2-intrastrand d(GpG)-cisplatin crosslink. Furthermore, placement of non-complementary thymine residues opposite the platinated guanines of a single 1,2-GG crosslink increases the efficiency of nucleotide excision repair of the adduct. These results argue that the specific structural alterations of the helix caused by intrastrand cisplatin-DNA crosslinks are the primary determinants of damage recognition and repair efficiency for the human NER machinery. Furthermore, the processing of 1,2-intrastrand cisplatin-DNA crosslinks in cells by mutagenic translesion DNA replication may modulate the extent to which 1,2-intrastrand cisplatin-DNA crosslinks are removed from DNA. Improved repair of a cisplatin crosslink after misincorporation of a base opposite may facilitate fixation of the mutation in some instances.

481 Nucleic Acids Acids Research, Research,1994, 1997,Vol. Vol.22, 25,No. No.13 Nucleic MATERIALS AND METHODS Human cell extracts, fractionated extracts and purified repair proteins Whole cell extracts were prepared as described previously (15) from the following cell lines: HeLa S3, CHO-9, GM2249 (XP-C), GM2485 (XP-D), CHO 27-1 (XP-B/ERCC3). Fractionation of HeLa whole cell extracts and preparation of purified XPA, TFIIH (Heparin-5PW fraction) and XPC-HHR23B proteins was as described previously (16–18). Construction of closed circular DNA containing single DNA lesions Oligonucleotides containing a single 1,3-intrastrand d(GpTpG)-cisplatin crosslink, 1,2-intrastrand d(GpG)-cisplatin crosslink and a single 2-acetylaminofluorene adduct were prepared as described previously (6,7,19). The 25mer oligonucleotide 5′-TCTTCTTCTCTAGTACTCTTCTTCT-3′ containing a 1,2intrastrand d(ApG)-cisplatin crosslink was synthesized in a similar way and was a kind gift of K. J. Yarema and J. M. Essigmann (MIT). Covalently closed circular DNA containing a single 1,3-intrastrand d(GpTpG) cisplatin crosslink (Pt-GTG or Pt-GTGx), a single 1–2-intrastrand d(GpG)-cisplatin crosslink (Pt-GG), a single 1,2-intrastrand d(ApG)-cisplatin crosslink (Pt-AG) and a single 2-acetylaminofluorene (AAF-G) adduct were produced by priming the plus strand of M13mp18GTG (20) or M13mp18GTGx (7), M13mp18GG (6), M13mp18AG and M13mp18AAF (19) respectively, with appropriate single lesion oligonucleotides as described (6,7,19). Control DNA substrates (Con-GTG or Con-GTGx, Con-GG, Con-AG and Con-AAF) were produced by the same method using the appropriate non-damaged oligonucleotides. The vector M13mp18AG was constructed by replacing the 38 base pair (bp) EcoRI–SalI fragment of M13mp18 with a sequence formed by annealing the oligonucleotides 5′-AATTCCTGGAGAAGAGAGTACTAGAGAAGAAGACCTGG-3′ and 5′-TCGACCAGGTCTTCTTCTCTAGTACTCTTCTTCTCCAGG-3′. Covalently closed circular DNA containing a single 1,2-intrastrand d(GpG)-cisplatin crosslink opposite either TpC (Pt-GG.TC), TpT (Pt-GG.TT) or CpT (Pt-GG.CT) in the direction 3′ to 5′ on the non-damaged DNA strand were produced as described above using the plus strand of M13mp18GG.TC, M13mp18GG.TT and M13mp18GG.CT respectively. These M13 vectors were formed by replacing the 179 bp EcoRI–PvuI restriction fragment of M13mp18GG with DNA duplexes obtained by PCR amplification of the replicative form of M13mp18GG using the primers 5′-AACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTAT-3′ and either 5′-CCATGATTACGAATTCCTGGAGAAGAAGAAGGCTTAGAAG-3′ (for M13mp18GG.TC), 5′-CCATGATTACGAATTCCTGGAGAAGAAGAAGGTTTAGAAG-3′ (for M13mp18GG.TT) or 5′-CCATGATTACGAATTCCTGGAGAAGAAGAAGGTCTAGAAG-3′ (for M13mp18GG.CT).

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of dATP, dCTP, dGTP and TTP, 40 mM phosphocreatine (di-Tris salt), 2.5 µg creatine phosphokinase (type I, Sigma), 3.4% glycerol and 18 µg bovine serum albumin. Cell extract was preincubated with buffer at 30C for 5 min, DNA substrate added, and reactions incubated at 30C for the times indicated. For repair synthesis assays 2 µCi [α-32P]dATP (3000 Ci/ mmol), 2 µCi [α-32P]dCTP (3000 Ci/mmol) and 2 µCi [α-32P]TTP (3000 Ci/mmol) were added to reaction mixtures. Purified DNA was digested in a 30 µl volume with 15 U BstNI (New England Biolabs) at 60C for 4 h prior to electrophoresis in non-denaturing 12% polyacrylamide gels. Phosphorimager (Molecular Dynamics) counts were used to calculate repair synthesis, taking account of the base composition of the damaged DNA strand of each restriction fragment. Incision assays using Pt-GTGx DNA were as described previously (7,22). Briefly, DNA purified from incision reactions was cleaved with HindIII and XhoI prior to separation on denaturing 12% polyacrylamide gels. DNA was then transferred to a nylon membrane (Hybond-N+, Amersham) by capillary transfer for 90 min. Fixed membranes were incubated for 16 h at 42C in hybridisation buffer containing 7% SDS, 10% PEG 8000, 250 mM NaCl, 130 mM potassium phosphate buffer (pH 7.0) and 100 pmol of the 27mer oligonucleotide 5′-GAAGAGTGCACAGAAGAAGAGGCCTGG-3′ labelled with [32P] at the 5′ terminus. Membranes were washed twice for 10 min in 1× SSC, 0.1% SDS before exposure to X-ray film with intensifying screens or a phosphorimager screen. Incision assays using Pt-GG DNA (and derivatives Pt-GG.TC, Pt-GG.TT and Pt-GG.CT containing mispaired thymines on the non-damaged DNA strand) were performed as described above except that purified DNA was incised with HindIII and EcoRI prior to denaturing gel electrophoresis and hybridisation was performed using a 32P-labelled 25mer oligonucleotide 5′-GAAGAAGGCCTAGAAGAAGACCTGG-3′ (Probe 1) complementary to the sequence of the damaged DNA strand flanking the 1,2-intrastrand d(GpG)-cisplatin crosslink. Analysis of the nondamaged DNA strand of Pt-GG DNA substrates was performed using a 32P-labelled 24mer oligonucleotide 5′-TCTTCTTCTAGGCCTTCTTCTTCT-3′ (Probe 2). Treatment with UvrABC endonuclease Purified Escherichia coli UvrA, UvrB, and UvrC proteins were used as described (6) to incise damaged DNA at the site of the lesion. For pre-incubation with UvrABC, 100 ng DNA was incubated in reaction buffer (in the absence of cell extract) for 15 min at 30C with 36 nM UvrA and 29 nM UvrB. UvrC was then added to 3.4 nM and the mixture was incubated for an additional 15 min. Finally, 200 µg of cell extract protein was added and the reactions were incubated for 3 h at 30C. Alternatively, the same amounts of UvrA, UvrB and UvrC proteins were added after 15 min pre-incubation with the cell extract. RESULTS

In vitro incision and repair synthesis reactions

Differential repair of single intrastrand cisplatin-DNA crosslinks

Reaction mixtures (50 µl, or multiples thereof) contained 100–300 ng of the appropriate single lesion or control DNA substrate and 100–200 µg whole cell extract (15) in a buffer containing 45 mM HEPES-KOH (pH 7.8), 70 mM KCl, 7.5 mM MgCl2, 0.9 mM DTT, 0.4 mM EDTA, 2 mM ATP, 5–20 µM each

Circular duplex DNA substrates containing a single 1,2-intrastrand d(GpG)-cisplatin crosslink (Pt-GG), a single 1,2-intrastrand d(ApG)-cisplatin crosslink (Pt-AG), a single 1,3-intrastrand d(GpTpG)-cisplatin crosslink (Pt-GTG) or a single 2-acetylaminofluorene adduct (AAF-G) were constructed (Fig. 1a). Each

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adduct is located in a unique restriction endonuclease target site and resistance to cleavage by the appropriate restriction enzyme is diagnostic for the presence of the adduct (7,23). DNA substrates containing single lesions were refractory to cleavage by the relevant enzyme (Fig. 1b). The small amount of nicked circular and linear DNA formed during these reactions indicates either that some unmodified DNA is present in the preparations, or that a restriction enzyme is occasionally able to cleave one or both DNA strands at the site of the adduct. This background nicking was most evident after incubation of DNA containing a single 1,2-intrastrand d(ApG)-cisplatin crosslink with ScaI. This is the only case where one of the phosphodiester bonds cleaved by the restriction enzyme is not immediately adjacent to an adducted base (Fig. 1a), which probably explains why this particular modified DNA is cleaved more efficiently. Control substrates lacking adducts were completely linearised by the diagnostic restriction endonucleases. Both adducted and nonmodified DNA substrates were completely linearised by SalI at a target site ∼15 bp away from the lesions (Fig. 1b). The presence of a single lesion on each DNA substrate was further confirmed by primer extension analysis of the damaged DNA strand [data not shown, see (7)]. Repair synthesis by human cell extracts at the site of the single adducts can be monitored by the incorporation of radiolabelled dNMPs into DNA spanning the site of the adduct. BstNI digestion of each substrate cuts the 500 bp region flanking each adduct into six fragments (Fig. 2a). The lesion is situated near the centre of a 29, 31 or 33 bp fragment (depending on the adduct) and is flanked by 68 bp and 99 or 100 bp fragments on the 5′ side and 3′ sides respectively. The remaining three BstNI restriction fragments contain 93% of the M13 DNA and are well separated from the site of damage. Similar levels of damage-dependent repair synthesis were detected in the 31 bp BstNI restriction fragment of AAF-G DNA and the 33 bp restriction fragment of Pt-GTG DNA (Fig. 2b, lanes 1–8). In contrast, much less repair synthesis arose with Pt-GG DNA (Fig. 2a, lanes 9–12) in accordance with our previous conclusion that this crosslink at this site is very poorly repaired by cell extracts (6). The Pt-AG DNA substrate was also poorly repaired (Fig. 2b lanes 13 and 14). We found that the addition of a nonionic detergent to reactions (in amounts near the critical micelle concentration) moderately stimulated repair synthesis and facilitated measurement of the low signal associated with Pt-GG DNA (Fig. 2c). Several independent experiments showed that Pt-GTG DNA was repaired 15–20-fold more efficiently than Pt-GG DNA. Pt-GTGx DNA, with a 3′ flanking DNA sequence somewhat different from Pt-GTG DNA, was also repaired 15–20-fold more efficiently than Pt-GG DNA (Fig. 8b). Absence of evidence for shielding of 1,2-intrastrand cisplatin-DNA crosslinks from repair enzymes by cellular proteins It has been proposed that 1,2-intrastrand cisplatin-DNA crosslinks are poorly repaired because they may be shielded from repair enzymes by cellular proteins which can bind to these adducts (6,12,24). Fractionation of human cell extracts should deplete or remove putative shielding proteins. We tested whether the use of fractions instead of whole cell extracts would increase the repair efficiency of Pt-GG DNA relative to Pt-GTG DNA. HeLa whole cell extract was fractionated (Fig. 3a) as described

Figure 1. Circular duplex DNA substrates containing single adducts. (a) The local DNA sequence flanking a single 1,3-intrastrand d(GpTpG)-cisplatin crosslink (Pt-GTG), a 1,2-intrastrand d(GpG)-cisplatin crosslink (Pt-GG), a 1,2-intrastrand d(ApG)-cisplatin crosslink (Pt-AG) and a 2-acetylaminofluorene adduct (AAF-G) within modified M13mp18 DNA are shown. Cleavage sites are indicated for SalI and for restriction endonucleases with target sites at each lesion. (b) Confirmation of the presence of site-specific DNA lesions. DNA substrates (Pt-GTG, Pt-GG, Pt-AG and AAF-G) or the corresponding non-modified control DNA duplexes (Con-GTG, Con-GG, Con-AG and Con-G respectively) were either untreated (U), incubated with SalI (S) or incubated with the restriction endonuclease having a recognition sequence encompassing the lesion (D; see a). After cleavage, products were separated by electrophoresis on a 1% agarose gel containing ethidium bromide, to separate covalently closed circular DNA (ccc), nicked circular DNA (nc), and linear DNA (lin).

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Figure 2. Differential repair synthesis at the site of adducts. (a) Sites of BstNI restriction cleavage are indicated by vertical bars and fragment lengths shown in bp. Six fragments are in a region of ∼500 bp region encompassing each adduct and three larger fragments are shown at the bottom. An AvaI site is located ∼430 bp 3′ to each single lesion. (b) Repair synthesis by human cell extracts at the site of single adducts is monitored by the incorporation of [32P] dNMPs into BstNI restriction fragments spanning the site of the adduct. Single lesion or control DNA substrates were incubated with HeLa cell extract for 3 h at 30C as described in Materials and Methods. Purified DNA was digested with BstNI, separated on a non-denaturing 12% polyacrylamide gel, fixed, dried and exposed to X-ray film or a phosphorimager screen. Lanes 1 and 2, 3 and 4, 5 and 6, 7 and 8, 9 and 10, and 11 and 12 are from duplicate reactions. The smallestBstNI restriction fragment in lanes 13 and 14 is observed as two bands probably due to some denaturation of this shorter 29 bp DNA duplex during electrophoresis. (c) Repair synthesis reaction mixture containing 0.01% Nonidet-P40, which stimulates the reaction enough to allow quantification of DNA synthesis associated with repair of Pt-GG DNA. The graph shows incorporation of [32P]dNMPs, corrected to the base composition of each restriction fragment.

previously (17) and Pt-GTG or Pt-GG DNA were incubated with various combinations of cellular fractions and purified repair proteins (Fig. 3b). We previously showed that fractions IIa, IIc, and IV were unnecessary for repair, that fraction I (FI) could be replaced with purified RPA and PCNA proteins, and that fraction IId could be replaced with purified XPG protein (17). However, the poor repair of Pt-GG DNA relative to Pt-GTG DNA by HeLa whole cell extract (lanes 1 and 2) did not improve after completely

omitting either FI (lanes 3 and 4) by replacement with RPA and PCNA, FI and FIV (lanes 9 and 10), IIa and FIV (lanes 15 and 16) or IIa, FIV and IId (lanes 17 and 18) by replacing IId with XPG. The difference in repair efficiency between Pt-GTG and Pt-GG remained at least 15-fold in all combinations tested. It is possible that some shielding proteins were present in Fractions IIb or III, but these results make it more likely that the repair of the 1,2-intrastrand cisplatin-DNA crosslink is inefficient because it is

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Figure 3. Comparison of repair of single 1,2-intrastrand d(GpG)-cisplatin and 1,3-intrastrand d(GpTpG)-cisplatin crosslinks by cellular fractions and purified proteins. (a) Fractionation scheme for HeLa whole cell extract, after ref. (17). The location of relevant repair proteins is indicated under the appropriate fractions. CFII was obtained by a 1 M KCl elution of proteins after whole cell extract proteins had been loaded onto phosphocellulose in 0.1 M KCl (56) and thus is equivalent to a combination of FII, FIII and FIV. (b) Repair synthesis experiment (similar to Fig. 2b) showing that omission of various fractions of HeLa cell extract and their replacement by purified repair proteins did not improve the poor repair of Pt-GG relative to Pt-GTG DNA.

an intrinsically poorer substrate for human nucleotide excision repair proteins. To investigate this point further, we used Escherichia coli UvrABC endonuclease to recognise and incise the single lesion DNA substrates prior to, or during, incubation with a human cell extract. Cell extracts were previously shown to be able to complete repair initiated by UvrABC at the site of a 1,2-intrastrand d(GpG)-cisplatin crosslink (6). For these experiments, we used cell extracts defective in the damage recognition protein XPA, to eliminate competing incision by the human repair complex. UvrABC endonuclease was added to DNA substrates prior to the addition of the XP-A cell extract, to allow UvrABC to recognise lesions and initiate incisions without any interference from human cell proteins, or added after incubating the XP-A cell extract with DNA, giving some time for any possible adductbinding proteins to shield DNA lesions. As expected, the XP-A cell extract was defective in repair of all four single lesions tested (Fig. 4, lanes 1–6). However, both pre-incision of the damaged DNA substrates with UvrABC (Fig. 4, lanes 7–12) and the addition of UvrABC after XP-A cell extract (Fig. 4, lanes 13–18) stimulated levels of repair synthesis to similar extents. The relative repair synthesis initiated by UvrABC for the four different single lesions had the order (AAF-G > Pt-GTG > Pt-GG > Pt-AG) similar to that catalyzed by human cell extracts in Figure 2 (AAF-G, Pt-GTG >> Pt-GG, Pt-AG). Because UvrABC recognised and incised lesions with similar efficiency in the presence or absence of cell extract, this shows that any cisplatin adduct binding proteins in the extract had no significant shielding effect.

Differential repair of 1,3- and 1,2-intrastrand cisplatin-DNA crosslinks is mediated during the recognition-incision stage of nucleotide excision repair To obtain an independent measure of the repair efficiency of Pt-GTG and Pt-GG DNA, the extent of dual incisions occuring at each of these lesions was measured by the formation of platinated oligonucleotides (Fig. 5). Dual incision of Pt-GTG DNA produced a characteristic pattern (7) of 24–30mer platinated oligonucleotides (Fig. 5, lane 3). To obtain a comparable signal intensity from the oligonucleotides formed during dual incision of Pt-GG DNA, it was found necessary to pool the products from 20 incision reactions (Fig. 5, lane 1). Purified DNA from one incision reaction is shown for Pt-GTG DNA (Fig. 5, lane 3). The formation of 24–30mer platinated oligonucleotides containing the 1,2-intrastrand d(GpG)-cisplatin crosslink was ∼15-fold less efficient than observed for the 1,3-intrastrand d(GpTpG)-cisplatin crosslink, consistent with the difference in repair synthesis measured for these two lesions. Improved repair of mispaired Pt-GG DNA substrates Structural studies (25–28) provide good evidence that the 1,3-GTG lesion distorts duplex DNA more than a 1,2-GG adduct. This distortion includes significant unwinding of the DNA helix, by 23 for a 1,3-intrastrand d(GpTpG)-cisplatin crosslink in comparison to 13 for a 1,2-intrastrand d(GpG)-cisplatin crosslink (25). We postulated that the placement of mispaired thymines on the non-damaged DNA strand directly opposite the platinated

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Figure 4. Cellular platinum adduct binding proteins do not prevent access of Escherichia coli UvrABC endonuclease to DNA lesions. Lanes 1–6, single lesion DNA substrates incubated with XP-A cell extract. Lanes 7–12, single lesion substrates incubated with UvrABC and then XP-A cell extract added and incubation continued for 3 h. Lanes 13–18, single lesion substrates incubated with XP-A cell extract for 15 min and then UvrABC added to the reaction mixture and incubation continued for 3 h. Repair synthesis initiated by UvrABC is largely contained within the smallest BstNI fragments of the four single lesion DNA substrates consistent with UvrABC making asymmetric incisions around each lesion 12–13 bases apart (7,57,58). Some damage-dependent synthesis also occurs in the 99 and 100 bp BstNI restriction fragments beyond the predicted 3′ incision site presumably because the UvrABC dual incision reaction is not well-coupled to the human DNA polymerase-mediated gap-filling.

guanines of a 1,2-intrastrand cisplatin-DNA crosslink might lead to increased structural distortion and improve the nucleotide excision repair efficiency of this DNA lesion. Three additional circular duplex M13 DNA substrates were synthesized, containing a single 1,2-intrastrand d(GpG)-cisplatin crosslink but with thymines in the non-damaged DNA strand opposite either the 5′ platinated guanine (Pt-GG.TC), the 3′ platinated guanine (Pt-GG.CT), or both platinated guanines (Pt-GG.TT) (Fig. 6a). Incubation of the mispaired Pt-GG DNA substrates with HeLa cell extract generated incision products of the same size range and pattern as was observed for Pt-GG DNA in Figure 5 (Fig. 6b, left, lanes 3, 5 and 7) with 27mers predominating. However, repair of the mismatched crosslinks was substantially more efficient than repair of the Pt-GG DNA substrate. A similar result was observed using CHO-9 cell extract (Fig. 6c). Weaker bands with a mobility corresponding to DNA fragments 43–46 nt long were also observed during repair of Pt-GG.TC, Pt-GG.TT and Pt-GG.CT DNA (Fig. 6c, lanes 3, 4, 5 and Fig. 7) and these are likely to be uncoupled 3′ incisions (22). Uncoupled incision during repair of Pt-GG DNA was below the

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Figure 5. Formation of dual incisions around single intrastrand cisplatin-DNA crosslinks. Pt-GG DNA from 20 incision reactions was pooled to obtain the excision products observed in lane 1. The predominant incision products were 27mers, as determined by comparison with a 24mer oligonucleotide 5′-TCTTCTTCTAGGCCTTCTTCTTCT-3′ (M1, lane 2) having a 5′-phosphate and a single 1,2-intrastrand d(GpG)-cisplatin crosslink bridging bases 11 and 12. The characteristic pattern of platinated oligonucleotides observed for Pt-GTG (7) (lane 3) was obtained using DNA from only one incision reaction. The predominant platinated oligonucleotides formed were 26mers, as determined by comparison with a 24mer oligonucleotide 5′-TCTTCTTCTGTGCACTCTTCTTCT-3′ (M2, lane 4) having a 5′-phosphate and a single 1,3-intrastrand d(GpTpG)-cisplatin crosslink bridging bases 10 and 12.

level of detection (Fig. 6b, left, lane 1; Fig. 6c, lane 2). The membrane shown at the left in Figure 6b was stripped and reprobed using an oligonucleotide complementary to 24 nt of the non-damaged DNA strand flanking the G.T mispairs (Fig. 6b, right). The 56 nt restriction fragment representing non-incised DNA produced a strong signal but no bands corresponding to incision of the non-damaged DNA strand were detected (lanes 1–8). This shows that the mispaired Pt-GG DNA substrates are significantly incised on the damaged DNA strand only. Excision products were not observed on either strand of unplatinated DNA containing a single or two adjacent G.T mispairs (Fig. 6b, lanes 4, 6 and 8) indicating that these mismatches are not good substrates for human NER. Cleavage of the damaged DNA strand of the mispaired Pt-GG DNA substrates was dependent on XP proteins, and reflected the normal nucleotide excision repair mechanism (Fig. 7). It was of particular interest to test XPC protein dependence, because it was recently found that at least one DNA alteration (a specific cholesterol moiety) could be repaired by NER in the absence of XPC (29). No incision products were observed, however, when the mismatched platinated DNA substrates were incubated with XP-C cell extract alone (Fig. 7, lanes 2, 4 and 6). Complementation of XP-C cell extract with purified XPC protein (Fig. 7, lanes 3, 5 and 7) restored the characteristic pattern of products, and the efficiency of repair of Pt-GG DNA remained low (lane 1). We also investigated whether the repair of Pt-GG.TT DNA was dependent on the presence of XPB and XPD, subunits of the TFIIH complex that is also required during the incision reaction. It was possible that the presence of two unpaired bases opposite the cisplatin-DNA adduct would provide an intrinsically more open or unwound DNA structure which might partially or completely alleviate the need for one or both of the helicase

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Figure 6. Improved incision of a 1,2-intrastrand d(GpG)-cisplatin crosslink when mispaired. (a) DNA substrates Pt-GG.TC, Pt-GG.TT and Pt-GG.CT containing a single 1,2-intrastrand d(GpG)-cisplatin crosslink opposite one or two mispaired thymine bases. The Pt-GTG and Pt-GG DNA substrates containing cytosines opposite the platinated guanines are shown for comparison. The oligonucleotide used as a primer during construction is underlined, and an x indicates the position of ligation to create circular duplexes. (b) The paired and mispaired substrates shown in (a), and their non-platinated controls, were incubated with HeLa cell extract for 30 min. Left, dual and uncoupled incisions were detected by hybridisation with Probe 1 as indicated in the diagram at the top. A characteristic pattern of platinated oligonucleotides was observed for Pt-GG.TC, Pt-GG.TT and Pt-GG.CT DNA (lanes 3, 5 and 7) with 27mers predominating. Platinated oligonucleotides are not visible for Pt-GG DNA in lane 1, which represents a single 50 µl reaction in contrast to the 20-fold reaction shown in Figure 5. The 29mer DNA fragment marked ‘x’ in lanes 2, 4, 6 and 8 represents a specific nick at the 11th phosphodiester bond 5′ to the lesion in a small fraction of the DNA substrate prior to incubation with human cell extracts, resulting from incomplete ligation of the synthesised complementary M13 DNA strand to the primer during substrate preparation (see a and text). Right, the membrane was stripped and hybridised with probe 2, complementary to the non-platinated DNA strand. (c) Dual incision products and uncoupled 3′ incisions formed by repair of Pt-GG.TC, Pt-GG.TT and Pt-GG.CT DNA by CHO-9 cell extract.

activities associated with TFIIH prior to the dual incision reaction. However, both XP-B and XP-D cell extracts were deficient in forming dual incisions around this lesion (lanes 8 and 10), and complementation of both extracts with purified TFIIH (lanes 9 and 11) restored the characteristic pattern of excision products.

NER of 1,2-intrastrand cisplatin-DNA adducts by extracts from mismatch repair-defective cells Recent studies have shown that human proteins that bind mismatches in DNA also have some affinity for cisplatin-DNA adducts. A 1,2-intrastrand d(GpG)-cisplatin crosslink with com-

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Figure 7. Repair of 1,2-intrastrand d(GpG)-cisplatin crosslinks opposite mispaired thymine bases is dependent on XPC, XPB, and XPD. Reactions were performed with Pt-GG and the three mispaired substrates and dual incisions detected as in Figure 6, either with XP-C cell extract GM2249 alone (lanes 2, 4 and 6) or after complementation with purified XPC-HHR23B complex (lanes 3, 5 and 7). Lanes 8 and 9, reactions were performed with Pt-GG.TT and extract from XP-B cells (CHO 27–1) or lanes 10 and 11, from XP-D cells (GM2485). Lanes 9 and 11 also contained purified TFIIH (5 µl, Heparin-5PW fraction).

plementary cytosine bases on the non-damaged strand was bound by the hMutSα complex consisting of hMSH2 and hMSH6/GTBP (30), and hMSH2 alone may also recognise this adduct (31). A direct comparison among several mismatched platinated molecules shows that hMutSα recognises duplex DNA containing a 1,2-intrastrand d(GpG)-cisplatin crosslink with much higher affinity when the non-damaged DNA strand contains a mispaired thymine opposite the 3′ platinated guanine (32). To test whether extracts defective in hMutSα have altered NER of such adducts, we investigated repair of normally paired and mispaired 1,2-intrastrand d(GpG)-cisplatin crosslinks by extracts from DLD-1 and LoVo cells, which are defective in the hMSH6/GTBP and hMSH2 subunits of hMutSα, respectively. A dual incision assay (Fig. 8a) showed that HeLa, DLD-1 and LoVo extracts incised Pt-GG DNA poorly (lanes 1, 5 and 9), but that Pt-GG.TC, Pt-GG.TT and Pt-GG.CT DNA substrates were incised significantly more efficiently (lanes 2–4, 6–8 and 10–12). Pt-GTGx DNA was incised with an efficiency similar to the mispaired substrates by all three extracts (data not shown). Only small and non-systematic differences (2–5-fold) were observed in the efficiency of dual incisions in the three mispaired substrates. These varied from 2- to 6-fold in further experiments using two separate preparations of each platinated DNA substrate (data not shown; see also Fig. 8c, lanes 3–5). We conclude that one or two non-complementary T residues opposite the intrastrand cisplatin crosslink increases in vitro repair of this lesion, and that hMutSα is not involved in this effect. In separate experiments, extracts

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from the hMLH1-defective HCT116 cell line also showed increased NER of the mispaired substrates (data not shown). Repair of these DNA substrates by both HeLa (Fig. 8b) and LoVo cell extracts (Fig. 8c) was also analysed by monitoring repair synthesis. Damage dependent repair synthesis by HeLa cell extract in the small BstNI restriction fragment containing the adduct for Pt-GTGx and the three mispaired Pt-GG DNA substrates was >17 fold higher than that seen for Pt-GG DNA (Fig. 8b). The same relative efficiency of repair was observed for these DNA substrates in LoVo cell extract (Fig. 8c, lanes 1–5). We note that the background DNA synthesis in the flanking 57, 68, 99, 127 and 139 bp restriction fragments was less for both Pt-GG and Con-GG DNA relative to the DNA substrates containing mispairs (compare Fig. 8b lanes 2 and 7 with lanes 3–5 and 8–10 respectively). Moreover, reactions using LoVo cell extract showed considerably less synthesis in these fragments than HeLa cell extract (compare lanes 3–5 and lanes 8–10 in Fig. 8b and c). This G.T mismatch-dependent and hMSH2-dependent background synthesis might represent a low level of bidirectional mismatch repair synthesis (33) initated at nicks formed during incubation with cell extract. Some of the DNA synthesis observed in the 68 and 127 bp fragments results because a small proportion of each DNA substrate contains a specific nick on the damaged strand at the 11th phosphodiester bond 5′ to the platinated guanine due to incomplete ligation of the primer to the synthesised complementary M13 DNA strand during substrate preparation (see Fig. 6a). Agarose gel electrophoresis (not shown) revealed that the Con-GG.TC DNA substrate contained a higher proportion of such contaminating nicked circular DNA molecules than other preparations. This is responsible for the increased DNA synthesis in all BstNI restriction fragments when incubated with HeLa cell extract (Fig. 8b, lane 8) and for the increased intensity of the band (x) corresponding to the position of the specific nick (Fig. 6b, lane 4). DISCUSSION Differential nucleotide excision repair of 1,2 and 1,3-intrastrand cisplatin-DNA crosslinks is not due to shielding of lesions from repair enzymes The structural distortion caused by a particular lesion in DNA may be influenced by the local nucleotide sequence and the topology of the substrate. To minimise effects of sequence, three cisplatin-DNA adducts were built into a circular duplex within the same DNA sequence except for a 6 bp region encompassing the adduct (Fig. 1a). We found by measuring repair synthesis that a 1,2-intrastrand d(GpG) or d(ApG)-cisplatin crosslink was repaired by HeLa cell extract 15–20-fold less efficiently than a 1,3-intrastrand d(GpTpG)-cisplatin crosslink in this sequence context. A similar difference of repair efficiency between the 1,2 and 1,3 crosslinks was found by measuring the formation of 3′ and 5′ incisions. Thus, the differential NER efficiency of these lesions is mediated by reaction steps preceding or coincident with endonucleolytic cleavage. The possibility that cellular proteins may shield 1,2-intrastrand cisplatin-DNA crosslinks from NER enzymes has been raised previously (6,12,24). Cellular factors that bind to the structural distortions caused by 1,2-intrastrand d(GpG)- and d(ApG)-cisplatin crosslinks have been detected in human cell extracts (12,34–37), notably several proteins containing HMG-box motifs (38–41). None of the HMG-box proteins tested were able

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Figure 8. Repair of 1,2-intrastrand d(GpG)-cisplatin crosslinks opposite mispaired thymine bases by cell extracts defective in hMSH2α. (a) Reactions were performed and dual incisions detected as in Figure 6, either with HeLa cell extract, hMSH6 defective DLD-1 cell extract, or hMSH2-defective LoVo cell extract as indicated. (b) Reactions performed with HeLa cell extract and repair synthesis detected as in Figure 2. (c) Reactions performed with LoVo cell extract and repair synthesis detected as in (b).

to bind a 1,3-intrastrand d(GpTpG)-cisplatin crosslink. Addition of a large excess of HMG protein to repair reactions can inhibit repair of a 1,2 but not a 1,3 crosslink (24). This effect might be caused by direct shielding, or by HMG-induced changes that make the DNA structure less favourable for repair by human NER (3,42). However, in the experiments reported here we found no evidence that proteins at normal levels in whole cell extracts play any significant role in shielding 1,2 crosslinks from repair. Use of

fractionated cell extracts and purified proteins (to deplete or remove putative cisplatin adduct binding factors) did not change the relative repair of 1,2 and 1,3 crosslinks. Moreover, incision of either type of crosslink by E.coli UvrABC repair endonuclease was not blocked by cell extract proteins. This conclusion is consistent with a recent report from Zamble et al. (8) that 1,2-cisplatin crosslinks are less efficiently repaired by cell extracts than 1,3-crosslinks and that the relative repair of these lesions is unchanged even when purified NER proteins are used in the dual incision reaction. Huang et al. (24) originally reported that both 1,2 and 1,3 crosslinks were well repaired, but the later study (8) concluded that the 1,3 crosslink was consistently repaired about 3-fold better than the 1,2 crosslink. We found a larger difference (15–20-fold) in the present experiments but there are some differences between the design of the studies. Apart from a 12 bp core, the DNA sequences flanking the lesions are dissimilar. Moreover, Zamble et al. used linear 156 bp DNA as substrate while we used 7.3 kbp closed circular DNA. We tested the effect of linearizing Pt-GTG and Pt-GG DNA with AvaI (Fig. 2a) prior to incubation with HeLa cell extract, but found no significant difference in repair efficiency compared to circular DNA (data not shown). This suggests that the topology of M13 duplex DNA does not strongly influence the differential repair of these lesions, although the length of flanking DNA sequences may be important. Calsou et al. also concluded that 1,2

489 Nucleic Acids Acids Research, Research,1994, 1997,Vol. Vol.22, 25,No. No.13 Nucleic crosslinks in cisplatin-damaged DNA are less well repaired than other less abundant adducts (9).

Increased NER of DNA adducts containing a mismatch Our data support the most likely explanation for the better repair of 1,3 over 1,2 cisplatin crosslinks, which is that the former adducts are better recognized by the human NER system because they cause more distortion of the DNA structure. By analogy, amongst UV-induced DNA damage products the more distorting (6–4) photoproducts are repaired ∼10-fold better than cyclobutane pyrimidine dimers (19), a difference that parallels the high discrimination of the damage-binding protein XPA for (6–4) photoproducts over cyclobutane dimers (16). The effect of further structural alteration at the site of the lesion was analysed by modifying the non-damaged DNA strand to contain mispaired thymine residues opposite one or both of the platinated guanines of the 1,2-intrastrand d(GpG)-cisplatin crosslink. G.T mispairs cause a structural change of the DNA helix (43) and the mispairing of platinated guanines with thymine bases could lead to a greater structural distortion more favourable for NER damage recognition. Consistent with this, 1,2-intrastrand d(GpG)-cisplatin crosslinks were 17-fold more efficiently repaired by HeLa cell extracts when opposite one or two T residues instead of C residues. We previously proposed that the precise incision locations vary for different types of DNA lesions whilst the distance between the two incision sites remains relatively constant (7). A 1,3-intrastrand d(GpTpG) crosslink was incised predominantly at the 8th and 9th phosphodiester bonds 3′ and the 16th, 19th and 20th bonds 5′ to the lesion, releasing 24–32mers with 26mers as the most predominant product (7). We now find that the 1,2-intrastrand d(GpG)-cisplatin crosslink is repaired with a similar but distinct incision pattern that gives rise to 27mers as the predominant excision product. Primer extension mapping together with measurement of the sizes of platinated oligonucleotides representing uncoupled 3′ incisions has revealed several preferred sites of cleavage around Pt-GG.TC, Pt-GG.TT and Pt-GG.CT DNA (5th, 7th, 8th, 10th-12th phosphodiester bonds 3′ to the lesion and the 15th, 18th and 19th phosphodiester bonds 5′ to the lesion; data not shown). Thus, the incision positions around both the 1,2- and the 1,3-intrastrand cisplatin crosslinks analysed here are different from each other and from other lesions including a thymine dimer (44). The distance between the 3′ and 5′ incisions is, however, a constant factor for all of these types of damage. There are interesting implications of the fact that the incision pattern is the same for both the correctly base-paired and mispaired 1,2-intrastrand d(GpG)-cisplatin crosslinks. Firstly, the sequences flanking the 1,2-intrastrand d(GpG)-cisplatin crosslink are not intrinsically refractory to cleavage by the NER endonucleases. Moreover, this suggests that the positioning of the 3′ and 5′ structure-specific endonucleases, XPG and XPF-ERCC1 respectively on the damaged DNA strand, probably in an open intermediate (45), is not strongly dependent on the DNA sequence of the non-damaged strand opposite the DNA lesion. The incision positions may be determined by the nature of the DNA adduct itself, whilst particular structural alterations of the DNA helix caused by the adduct determine the efficiency of damage recognition. A precedent for this feature of damage

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recognition can be found in a recent study of E.coli UvrABC endonuclease in which the incision positions around a 2-acetylaminoflourene adduct did not change when present in either duplex DNA or DNA mimicking a slipped DNA intermediate in which the bases opposite the lesion are not complementary (46).

Improved nucleotide excision repair of DNA damage and mutagenic translesion DNA synthesis The finding that mispaired 1,2-intrastrand cisplatin-DNA crosslinks are removed more efficiently than the same DNA lesion opposite a complementary DNA strand may have implications for cisplatin-damage processing within cells. The 1,2-intrastrand d(GpG)-cisplatin crosslinks are the most abundant lesions formed both in vitro and in vivo by cisplatin (3). Many 1,2-intrastrand d(GpG)-cisplatin crosslinks are known to undergo replication bypass in cells treated with cisplatin (47,48). A recent study demonstrated that fork-like DNA templates containing cisplatin GG lesions undergo translesion DNA synthesis very efficiently in mammalian cell extracts (49). In cases where 1,2-intrastrand cisplatin-DNA crosslinks do undergo bypass with misincorporation of a base opposite one of the adducted guanines, our results suggest that the platinum crosslink will be more readily removed in a subsequent NER event. Repair synthesis to fill in the gap after excision of the platinated oligonucleotide will copy the former mismatch, fixing the mutation in both strands. The consequence is that nucleotide excision repair would promote mutagenesis in this situation. On the other hand, it is clear that NER normally acts to decrease mutation frequency in damaged DNA, because NER-defective mammalian cells show a much increased frequency of mutation by various DNA damaging agents (50,51), including cisplatin treatment (52). However, increased repair of mismatched adducts might significantly influence mutagenesis at particular sites that are efficiently bypassed in an error-prone manner. There are indications that the mismatch repair and NER pathways interact with one another in mammalian cells. The development of cisplatin resistance in some cases has been associated with specific defects in mismatch repair that result in microsatellite instability (53,54). A possible explanation is that binding of mismatch repair proteins to cisplatin lesions (30,31), particularly when opposite non-complementary bases (32), could interfere with NER. In this view, loss of mismatch repair proteins might improve NER and increase resistance to cisplatin. The present study shows that NER is in fact much more effective when a 1,2 cisplatin intrastrand crosslink is opposite one or two non-complementary thymines. The mechanism of this increased repair does not involve mismatch recognition proteins, because the same difference in NER was also found with extracts from mismatch repair-deficient cells. Nevertheless, active mismatch repair in cells could conceivably compete with NER if a mismatch occurs across from an adduct. The present experiments have not directly explored this possibility, but such studies would be feasible. In certain special instances, such as preferential repair of transcribed DNA strands, an interaction of the mismatch repair system and NER may even be cooperative. Mellon and coworkers (55) found defects in the removal of UV lesions from the transcribed strand of active genes in mismatch-repair defective cells. If an in vitro system is developed to simultaneously carry

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out transcription-coupled NER and mismatch repair, the relevant mechanism can be explored. ACKNOWLEDGEMENTS We are very grateful to L. Grossman (Johns Hopkins University) for the E.coli UvrABC proteins, J.-M. Egly (IGBMC, Strasbourg) for the TFIIH sample, and K. J. Yarema and J. M. Essigmann for providing some of the platinated oligonucleotides used in this study. We thank the members of our laboratory for help, Elizabeth Evans and Rafael Ariza for comments on the manuscript, and the ICRF Cell Production department for assistance. REFERENCES 1 Andrews, P.A. and Howell, S.B. (1990) Canc. Cells, 2, 35–43. 2 Timmer-Bosscha, H., Mulder, N. and de Vries, E. (1992) Br. J. Cancer, 66, 227–238. 3 Zamble, D.B. and Lippard, S.J. (1995) Trends Biochem. Sci., 20, 435–439. 4 Bedford, P., Fichtinger-Schepman, A.M.J., Shellard, S.A., Walker, M.C., Masters, J.R.W. and Hill, B.T. (1988) Cancer Res., 48, 3019–3024. 5 Eastman, A. and Schulte, N. (1988) Biochemistry, 27, 4730–4734. 6 Szymkowski, D.E., Yarema, K.J., Essigmann, J.E., Lippard, S.J. and Wood, R.D. (1992) Proc. Natl. Acad. Sci. USA, 89, 10772–10776. 7 Moggs, J.G., Yarema, K.J., Essigmann, J.M. and Wood, R.D. (1996) J. Biol. Chem., 271, 7177–7186. 8 Zamble, D.B., Mu, D., Reardon, J.T., Sancar, A. and Lippard, S.J. (1996) Biochemistry, 35, 10004–10013. 9 Calsou, P., Frit, P. and Salles, B. (1992) Nucleic Acids Res., 20, 6363–6368. 10 Wood, R.D. (1996) Annu. Rev. Biochem. 65, 135–167. 11 Sancar, A. (1996) Annu. Rev. Biochem., 65, 43–81. 12 Donahue, B.A., Augot, M., Bellon, S.F., Treiber, D.K., Toney, J.H., Lippard, S.J. and Essigmann, J.M. (1990) Biochemistry, 29, 5872–5880. 13 Treiber, D., Zhai, X., Jantzen, H. and Essigmann, J. (1994) Proc. Natl. Acad. Sci. USA, 91, 5672–5676. 14 Gunz, D., Hess, M.T. and Naegeli, H. (1996) J. Biol. Chem., 271, 25089–25098. 15 Wood, R.D., Biggerstaff, M. and Shivji, M.K.K. (1995) Methods, 7, 163–175. 16 Jones, C.J. and Wood, R.D. (1993) Biochemistry, 32, 12096–12104. 17 Aboussekhra, A., Biggerstaff, M., Shivji, M.K.K., Vilpo, J.A., Moncollin, V., Podust, V.N., Protic’, M., Hübscher, U., Egly, J.-M. and Wood, R.D. (1995) Cell, 80, 859–868. 18 Hwang, J.R., Moncollin, V., Vermeulen, W., Seroz, T., van Vuuren, H., Hoeijmakers, J.H.J. and Egly, J.M. (1996) J. Biol. Chem., 271, 15898–15904. 19 Szymkowski, D.E., Lawrence, C.W. and Wood, R.D. (1993) Proc. Natl. Acad. Sci. USA, 90, 9823–9827. 20 O’Donovan, A., Davies, A.A., Moggs, J.G., West, S.C. and Wood, R.D. (1994) Nature, 371, 432–435. 21 Szymkowski, D.E., Hajibagheri, M.A.N. and Wood, R.D. (1993) J. Mol. Biol., 231, 251–260. 22 Sijbers, A.M., de Laat, W.L., Ariza, R.R., Biggerstaff, M., Wei, Y.-F., Moggs, J.G., Carter, K.C., Shell, B.K., Evans, E., de Jong, M.C., Rademakers, S., de Rooij, J., Jaspers, N.G.J., Hoeijmakers, J.H.J. and Wood, R.D. (1996) Cell, 86, 811–822. 23 Naser, L.J., Pinto, A.L., Lippard, S.J. and Essigmann, J.M. (1988) Biochemistry, 27, 4357–4367. 24 Huang, J.C., Zamble, D.B., Reardon, J.T., Lippard, S.J. and Sancar, A. (1994) Proc. Natl. Acad. Sci. USA, 91, 10394–10398.

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