Human and E.coli excinucleases are affected differently by the sequence context of acetylaminofluorene-guanine adduct

Synthetic DNA substrates containing an acetylaminofluorene (AAF) adduct at each of the three guanine in the G1G2CG3CC sequence were constructed and tested as substrates for reconstituted E.coli (A)BC excinuclease and human excinuclease in HeLa cell-free extract (CFE). The (A)BC excinulcease repaired the three substrates with relative efficiencies of G1:G2:G3 of 100:18:66 in agreement with an earlier report [Seeberg, E., and Fuchs, R.P.P. (1990) Proc. Natl Acad. Sci. USA 87, 191-194]. The same lesions were repaired by the human excinuclease with the strikingly different efficiencies of G1:G2:G3 as 38:100:68. These results reveal that the human excinuclease is affected by the sequence context of the lesion in a different manner than its prokaryotic counterpart.     

Comparison of sequence preference of tomaymycin- and anthramycin-DNA bonding by exonuclease III and lambda exonuclease digestion and UvrABC nuclease incision analysis

The DNA bonding sites of two pyrrolo[1,4]benzodiazepine derivatives--tomaymycin (Tma) and anthramycin (Atm)--were identified by exonuclease III (exo III) digestion, lambda exonuclease (lambda exo) digestion, and UvrABC nuclease incision analysis. exo III digestion stalls 4-5 bases 3' to a drug-DNA adduct. While this method can recognize most of the Atm-and Tma-DNA modification sites, it is complicated in that exo III digestion is also stalled by certain unmodified sequences and by drug bound to the opposite strand. lambda exo digestion stalls 1-2 bases 5' to a drug-DNA adduct. The lambda exo method also recognizes most of the drug-DNA bonding sites and renders a cleaner background; however, it is also affected by opposite-strand drug bonding. Due to their intrinsic digestion polarities, these two exonucleases tend to be stalled by the drug-DNA adduct at one end of the DNA molecule. Purified UvrA, UvrB, and UvrC proteins acting together make dual incisions 6-8 bases 5' and 4 bases 3' to a Atm- or Tma-DNA adduct. This nuclease complex recognizes all the Tma- and Atm-DNA bonding sites identified by exonuclease digestion methods, and all the UvrABC incisions can be attributed to drug modifications in the incised DNA strand. The degree of UvrABC nuclease incision increases with increasing drug concentrations for DNA modification. Using the UvrABC incision method, we have identified the sequence preference of Tma- and Atm-DNA adduct formation in three DNA fragments, and we have found that these two drugs have different preferred sites for adduction. Both Tma- and Atm-DNA bonding is strongly influenced by the 5' and 3' neighboring bases; the orders of preferred 5' and 3' bases for Tma are A > G, T > C, and A, C > G, T, and for Atm the orders are A > G > T > C and A > G > T, C. The preferred triplets for Tma bonding are -AGA- > -GGC-, -TGC-, and AGC- and for Atm are -AGA-, -AGG- > -GGA-, -GGG-.     

A slipped replication intermediate model is stabilized by the syn orientation of N-2-aminofluorene- and N-2-(acetyl)aminofluorene-modified guanine at a mutational hotspot

The Escherichia coli NarI restriction enzyme recognition site 5'G1G2C3G4C5C63' is a mutational hotspot for -2 deletions in E. coli plasmid pBR322, resulting in the sequence 5'GGCC3' when G4 is modified by the aromatic amine N-2-(acetyl)aminofluorene (AAF) [Burnouf, D., Koehl, P., and Fuchs, R. P. P. (1995) Proc. Natl. Acad. Sci. U.S.A. 86, 4147-4151] even though each G shows similar reactivity [Fuchs, R. P. P. (1984) J. Mol. Biol. 177, 173-180]. Modification at G4 by the related aromatic amine 2-aminofluorene (AF), which lacks the acetyl group of AAF, can also cause -2 deletions, but at a lower frequency [Bichara, M., and Fuchs, R. P. P. (1985) J. Mol. Biol. 183, 341-351]. A specific mechanism has been proposed to explain the double-base frameshifts in the NarI sequence in which the GC deletion results from a slipped mutagenic intermediate formed during replication [Schaaper, B. M., Koffel-Schwartz, N., and Fuchs, R. P. P. (1990) Carcinogenesis 11, 1087-1095]. We address the following key questions in this study. Why does AAF modification dramatically increase the mutagenicity at the NarI G4 position, and why does AAF enhance the mutagenicity more than AF? We studied two intermediates which model replication at one arm of a fork, using a fragment of DNA modified by AF or AAF at G4 in the NarI sequence: Intermediate I can be converted into intermediate II by misalignment. Elongation of intermediate I leads to error-free translesion synthesis, while elongation of intermediate II leads to a -2 frameshift mutation. Minimized potential energy calculations were carried out using the molecular mechanics program DUPLEX to investigate the conformations of the AF and AAF adducts at G4 in these two intermediates. We find that the slipped mutagenic intermediate is quite stable relative to its normally extended counterpart in the presence of AF and AAF in an abnormal syn orientation of the damaged base. An enhanced probability of elongation from a stable slipped structure rather than a properly aligned one would favor increased -2 frameshift mutations. Furthermore, AAF-modified DNA has a greater tendency to adopt the syn orientation than AF because of its greater bulk, which could explain its greater propensity to cause -2 deletions in the NarI sequence.     

A molecular mechanics and dynamics study of the minor adduct between DNA and the carcinogen 2-(acetylamino)fluorene (dG-N2-AAF)

Experimental studies involving the carcinogenic aromatic amine 2-(acetylamino)fluorene (AAF) have afforded two acetylated DNA adducts, the major one bound to C8 of guanine and a minor adduct bound to N2 of guanine. The minor adduct may be important in carcinogenesis because it persists, while the major adduct is rapidly repaired. Primer extension studies of the minor adduct have indicated that it blocks DNA synthesis, with some bypass and misincorporation of adenine opposite the lesion [Shibutani, S., and Grollman, A.P. (1993) Chem. Res. Toxicol. 6, 819-824]. No experimental structural information is available for this adduct. Extensive minimized potential energy searches involving thousands of trials and molecular dynamics simulations were used to study the conformation of this adduct in three sequences: I, d(C1-G2-C3-[AAF]G4-C5-G6-C7).d(G8-C9-G10-C11-G12-C13-G14+ ++); II, the sequence of Shibutani and Grollman, d(C1-T2-A3-[AAF]G4-T5-C6-A7).d(T8-G9-A10-C11-T12-A13-G14); and III, which is the same as II but with a mismatched adenine in position 11, opposite the lesion. AAF was located in the minor groove in the low-energy structures of all sequences. In the lowest energy form of the C3-[AAF]G4-C5 sequence I, the fluorenyl rings point in the 3' direction along the modified strand and the acetyl in the 5' direction. These orientations are reversed in the second lowest energy structure of this sequence, and the energy of this structure is 1.4 kcal/mol higher. Watson Crick hydrogen bonding is intact in both structures. In the two lowest energy structures of the A3-[AAF]G4-T5 sequence II, the AAF is also located in the minor groove with Watson-Crick hydrogen bonding intact. However, in the lowest energy form, the fluorenyl rings point in the 5' direction and the acetyl in the 3' direction. The energy of the structure with opposite orientation is 5.1 kcal/mol higher. In sequence III with adenine mismatched to the modified guanine, the lowest energy form also had the fluorenyl rings oriented 5' in the minor groove with intact Watson-Crick base pairing. However, the mispaired adenine adopts a syn orientation with Hoogsteen pairing to the modified guanine. These results suggest that the orientation of the AAF in the minor groove may be DNA sequence dependent. Mobile aspects of favored structures derived from molecular dynamics simulations with explicit solvent and salt support the essentially undistorting nature of this lesion, which is in harmony with its persistence in mammalian systems.     

Mismatch-, MutS-, MutL-, and helicase II-dependent unwinding from the single-strand break of an incised heteroduplex

Escherichia coli MutS, MutL, and DNA helicase II are sufficient to initiate mismatch-dependent unwinding of an incised heteroduplex (Yamaguchi, M., Dao, V., and Modrich, P. (1998) J. Biol. Chem., 273, 9197-9201). We have studied unwinding of 6.4-kilobase circular G-T heteroduplexes that contain a single-strand incision, 808 base pairs 5' to the mismatch or 1023 base pairs 3' to the mispair as viewed along the shorter path between the two DNA sites. Unwinding of both substrates in the presence of MutS, MutL, DNA helicase II, and single-stranded DNA binding protein was mismatch-dependent and initiated at the single-strand break. Although unwinding occurred in both directions from the strand break, it was biased toward the shorter path linking the strand break and the mispair. MutS and MutL are thus sufficient to coordinate mismatch recognition to the orientation-dependent activation of helicase II unwinding at a single-strand break located a kilobase from the mispair.     

Binding of actinomycin D to DNA oligomers of CXG trinucleotide repeats

Actinomycin D (ACTD) binding propensities of DNA with CXG trinucleotide repeats were investigated using oligomers of the form d[AT(CXG)n = 2-4AT] and their corresponding heteroduplexes, where X = A, C, G, or T. These oligonucleotides contain -CXGCXG-, -CXGCXGCXG-, and -CXGCXGCXGCXG- units that can form homoduplexes containing one, two, and three GpC binding sites, respectively, with flanking X/X mismatches. The corresponding heteroduplexes contain these same sites with flanking Watson-Crick base pairs. It was found that oligomers with X = G exhibit weak ACTD affinities whereas those with X not equal to G and n = 3 exhibit unusually strong ACTD binding affinities with binding constants ranging from 2.3 x 10(7) to 3.3 x 10(7) M-1 and binding densities of approximately 1 drug molecule/strand (or 2/duplex). These binding affinities are considerably higher than those of their shorter and longer counterparts and are about 2- and 10-fold stronger than the corresponding CAG.CTG and CGG.CCG heteroduplexes, respectively. The CTG-containing oligomer d[AT(CTG)3AT] stands out as unique in having its ACTD dissociation kinetics being dominated by a strikingly slow process with a characteristic time of 205 min at 20 degrees C, which is 100-fold slower than d[AT(CAG)3AT], nearly 10-fold slower than the corresponding heteroduplex, and considerably slower than d[AT(CTG)2AT] (63 min) and d[AT(CTG)4AT] (16 min). The faster dissociation rate of the n = 4 oligomer compared to its n = 2 counterpart is in apparent contrast with the observed 10-fold stronger ACTD binding affinity of the former. It was also found that d[AT(CCG)3AT] exhibits the slowest dissociation rate of the CGG/CCG series, being more than an order of magnitude slower than that of its heteroduplex (tau slow of 43 vs 2 min). The finding that a homoduplex d[AT-CXG-CXG-CXG-AT]2 can bind two ACTD molecules tightly is significant since it was thought unlikely for two consecutive GpC sites separated by a single T/T mismatch to do so.     

Incision at DNA G.T mispairs by extracts of mammalian cells occurs preferentially at cytosine methylation sites and is not targeted by a separate G.T binding reaction

We have investigated the specificities of G.T mismatch binding proteins and of G.T mismatch cleavage in extracts of mammalian cells. G.T mismatch-specific protein:DNA complex formation by cell extracts was independent of the local sequence context of the mismatch. Cell extracts performed similar levels of protein binding to DNA substrates in which a single G.T mispair was preceded by T, G, A, C, or 5-meC. In contrast, incision by extracts of the T-containing strand of a G.T mismatch exhibited a strong sequence specificity and efficient strand cleavage was only observed when the mismatched G was in a CpG sequence. Thus, oligonucleotides containing either CpgGpT or 5meCpGGpT were efficiently incised, but not those containing GpGCpT, ApGTpT, or TpGApT sequences. Cell lines made resistant to the alkylating agent N-methyl-N-nitrosourea have previously been found to be defective in a G.T mismatch binding reaction. The defect in binding by extracts prepared from these cells extended to G.T mismatches in several sequence contexts. The variant extracts nevertheless incised G.T mismatches normally suggesting that this particular binding activity is not required for incision. The data indicate that incision by this activity is targeted to the CpG sequences in which G.T mismatches are formed by the mutagenic deamination of DNA 5-methylcytosine. In this regard the repair pathway resembles the very short patch (vsp) repair pathway in Escherichia coli.     

Strand- and sequence-specific attenuation of N-methyl-N'-nitro-N-nitrosoguanidine-induced G.C to A.T transitions by expression of human 6-methylguanine-DNA methyltransferase in Chinese hamster ovary cells

The effect of human O6-methylguanine-DNA methyltransferase (MGMT) on the cytotoxicity, the mutagenicity, and the specific kinds of base substitutions induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were examined in non-MGMT transfected Chinese hamster ovary cells (CHOM cells) and in those cells which had been transfected with human MGMT complementary DNA (AGT cells). AGT cells containing a high level of human MGMT activity were markedly more resistant to the cytotoxic and mutagenic effects of MNNG than CHOM cells which had no detectable MGMT activity. The dosages of MNNG which reduced to 50% of colony forming ability were estimated to be 0.8 microM for CHOM and 10 microM for AGT cells. The induction frequency of 6-thioguanine-resistant cells was significantly declined in AGT cells. At 4 microM MNNG, this frequency was declined from 273 mutants/10(6) viable CHOM cells to 13 mutants/10(6) viable AGT cells. The entire coding region of the hypoxanthine (guanine) phosphoribosyltransferase (hprt) gene in 37 AGT and 22 CHOM mutants was characterized by direct sequencing of the mRNA-polymerase chain reaction-amplified complementary DNA. Base changes at the intron-exon boundaries of the hprt DNA in the splicing mutants were further examined. Those results indicated that G to A transitions were significantly reduced in MNNG-treated AGT cells (chi 2 test, P < 0.001), suggesting that O6-methylguanine was repaired error free by human MGMT. In contrast, no difference arose in the frequencies of T to C transitions induced by MNNG in these two populations. All of the G to A transitions induced in AGT cells were located on the nontranscribed strand, assuming that the causative lesion was O6-methylguanine (P < 0.05). Such a strand specificity was not observed in CHOM mutants. Most of the G to A transitions observed in CHOM mutants were located at the middle guanine of 5'-GGPu sequences. Transitions observed at these sites, particularly 5'-GGG, were significantly reduced in AGT mutants (P < 0.05). Our results have suggested that human MGMT specifically repairs O6-methylguanine with a preference to remove those located on the transcribed strand and middle guanine of 5'-GGG.     

Effect of the prenatal maternal environment on the control of breathing during non-rapid-eye-movement sleep in the developing lamb

The specific recognition of DNA modifications by repair endonucleases was used to characterize damage induced by 3-carbethoxypsoralen (3-CPs) plus UvA in M13mp8 replicative form I (RF-I) DNA. Under the conditions used, 3-CPs plus UVA generates DNA base modifications which are recognized by the UvrABC complex and the Fpg protein of E. coli. The rate of formation of UvrABC sensitive sites is 3-4-fold higher than that of Fpg sensitive sites. In addition a small number of sites of base loss (sensitive to Nfo protein) were observed. M13mp8 RF-I DNA treated with 3-CPs plus UVA was tested for transfection efficiency in E. coli mutants defective in either Fpg protein and/or UvrABC complex. The survival of 3-CPs plus UVA damaged M13mp8 RF-I DNA was significantly reduced when transfected into uvrA mutants compared to that in the wild-type strain. On the other hand, the survival of 3-CPs plus UVA damaged RF-I DNA was not altered in fpg-1 mutants. These results show that nucleotide excision repair mediated by the UvrABC complex is the major repair pathway involved in the elimination of lethal lesions induced in DNA by 3-CPs plus UVA. Our data suggest that in vitro exposure of M13mp8 RF-I DNA to 3-CPs plus UVA produces predominantly thymine photoaddition and to a lesser extent guanine photooxidation partially due to singlet oxygen generated during photoreaction. The photoaddition products are primarly responsible for the observed lethal effect.     

Detection of p53 point mutations by double-gradient, denaturing gradient gel electrophoresis

Genetic instability is a typical feature of tumor cells. This evidence has stimulated the development of rapid methods for detection of gene mutations. A new, improved protocol for denaturing gradient gel electrophoresis (DGGE), to screen for point mutations in genomic DNA, is reported: double gradient (DG) DGGE. In this technique, to the primary, denaturing gradient (typically 30-80% or 40-80% urea/formamide) a secondary gradient, colinear with the first, is superimposed: a porosity gradient (typically 6.5-12% polyacrylamide). The secondary gradient acts by recompacting smeared and diffuse bands of heteroduplexes, which are often indistinguishable from background fluorescence, and by augmenting the resolution between closely spaced homoduplex zones. This allows proper densitometric quantitation of the ratio of the two homoduplex bands. The reliability of this technique has been documented by detection of a number of mutations in exons 6 and 8 of the p53 gene which had escaped revelation by single-strand conformational polymorphism (SSCP) analysis. Additionally, the precise assessment of ratio of the doublet of homoduplex bands has allowed quantitation of the extent of p53 mutation in a mixed cell population extracted from a tumor specimen.     

Human nucleotide excision nuclease incises synthetic double-stranded DNA containing a pyrimidine dimer at the fourth phosphodiester linkage 3' to the pyrimidine dimer

Linear 75mer double-stranded DNA containing a single pyrimidine dimer at a unique site was used to investigate pyrimidine dimer-dependent endonuclease activities from human cells. HeLaS3 cell extract incised the target DNA at the fourth phosphodiester linkage 3' to the pyrimidine dimer. However, incision of the DNA at 5' side of the pyrimidine dimer was not detected. The incision was also detected in cell extracts prepared from other excision repair-proficient cell lines. Incision was detected only on the DNA strand containing a pyrimidine dimer in the presence of poly(dI-dC)-poly(dI- dC) double strand. The reaction required Mg2+ but not ATP. The extract prepared from excision repair-deficient xeroderma pigmentosum (XP) cells belonging to the complementation group A was unable to incise the DNA. Extracts from the complementation groups C, D, and G incised the DNA very weakly at the third phosphodiester linkage 3' to the pyrimidine dimer, a site different from that incised by normal human cell extract. These results suggest that the observed incision reaction is associated with excision repair in human cells.     

Cellular strategies for accommodating replication-hindering adducts in DNA: control by the SOS response in Escherichia coli

The replication of double-stranded plasmids containing a single adduct was analyzed in vivo by means of a sequence heterology that marks the two DNA strands. The single adduct was located within the sequence heterology, making it possible to distinguish trans-lesion synthesis (TLS) events from damage avoidance events in which replication did not proceed through the lesion. When the SOS system of the host bacteria is not induced, the C8-guanine adduct formed by the carcinogen N-2-acetylaminofluorene (AAF) yields less than 1% of TLS events, showing that replication does not readily proceed through the lesion. In contrast, the deacetylated adduct N-(deoxyguanosin-8-yl)-2-aminofluorene yields approximately 70% of TLS events under both SOS-induced and uninduced conditions. These results for TLS in vivo are in good agreement with the observation that AAF blocks DNA replication in vitro, whereas aminofluorene does so only weakly. Induction of the SOS response causes an increase in TLS events through the AAF adduct (approximately 13%). The increase in TLS is accompanied by a proportional increase in the frequency of AAF-induced frameshift mutations. However, the polymerase frameshift error rate per TLS event was essentially constant throughout the SOS response. In an SOS-induced delta umuD/C strain, both US events and mutagenesis are totally abolished even though there is no decrease in plasmid survival. Error-free replication evidently proceeds efficiently by means of the damage avoidance pathway. We conclude that SOS mutagenesis results from increased TLS rather than from an increased frameshift error rate of the polymerase.     

Identification of the persistently bound form of the carcinogen N-acetyl-2-aminofluorene to rat liver DNA in vivo

In this article the structural analysis of the persistently bound form of the carcinogen N-acetyl-2-aminofluorene (AAF) to rat liver DNA in vivo is described. This compound appears to result from the formation of a covalent bond between carbon-3 of the aromatic ring and the amino group of guanine. Experimental evidence from three different approaches had led to the identification of the structure of the persistently DNA-bound AAF moiety. First, [3-3H, 9-14C]N-acetoxy-AAF was reacted with DNA in vitro. As reported previously, a minor product was isolated from enzymatic digests of the reacted DNA, which had chemical and chromatographic properties identical to those of the persistent--AAF moiety in DNA in vivo. The ratio 3H/14C of this product had diminished to the same extent as 3-CH3S-AAF resulting from the reaction of methionine with [o-3H, 9-14C]N-acetoxy-AAF. Secondly, reaction of [9-14C]N-acetoxy-AAF with DNA, which was tritiated in the C-8 positions of the purines, did not result in removal of tritium in the persistent fraction obtained after acid hydrolysis, thus excluding substitution at C-8 and N-7 of guanine. Finally , by reacting N-OSO3-K-AAF with deoxyguanosine in dimethylsulfoxide-triethylamine, a compound could be isolated, which was identified as 3-(deoxyguanosin-N2-yl)-AAF based on its NMR spectrum and on the mass spectrum of the corresponding guanine derivative obtained after removing deoxyribose by acid hydrolysis. This compound appeared to be identical with the persistently bound form present in DNA hydrolysates from rat liver after injection of [2'-3H]N-hydroxy-AAF.     

Helicase motifs V and VI of the Escherichia coli UvrB protein of the UvrABC endonuclease are essential for the formation of the preincision complex

The UvrB protein is a subunit of the UvrABC endonuclease which is involved in the repair of a large variety of DNA lesions. We have 91 isolated random uvrB mutants which are impaired in the repair of UV-damage in vivo. These mutants were classified on the basis of the ability to form normal levels of protein and the position of the mutations in the gene. The amino acid substitutions in the N-terminal part or in the C-terminal part of the UvrB protein are exclusively found in the conserved boxes of the so-called "helicase motifs" present in these parts of the protein, indicating that these motifs are essential for UvrB function. The proteins of four C-terminal mutants were purified: two mutants in motif V (E514K and G509S), one mutant in motif VI (R544H) and a double mutant in both motifs (E514K + R541H). In vitro experiments with these mutant proteins show that the helicase motifs V and VI are involved in the induction of ATP hydrolysis in the presence of (damaged) DNA and in the strand-displacement activity of the UvrA2B complex as is observed in a helicase assay. Furthermore, our results suggest that this strand-displacement activity is correlated to a local unwinding, which seems to be used to form the UvrB-DNA preincision complex.     

Slow repair of bulky DNA adducts along the nontranscribed strand of the human p53 gene may explain the strand bias of transversion mutations in cancers

Using UvrABC incision in combination with ligation-mediated PCR (LMPCR) we have previously shown that benzo(a)pyrene diol epoxide (BPDE) adduct formation along the nontranscribed strand of the human p53 gene is highly selective; the preferential binding sites coincide with the major mutation hotspots found in human lung cancers. Both sequence-dependent adduct formation and repair may contribute to these mutation hotspots in tumor tissues. To test this possibility, we have extended our previous studies by mapping the BPDE adduct distribution in the transcribed strand of the p53 gene and quantifying the rates of repair for individual damaged bases in exons 5, 7, and 8 for both DNA strands of this gene in normal human fibroblasts. We found that: (i) on both strands, BPDE adducts preferentially form at CpG sequences, and (ii) repair of BPDE adducts in the transcribed DNA strand is consistently faster than repair of adducts in the nontranscribed strand, while repair at the major damage hotspots (guanines at codons 157, 248 and 273) in the nontranscribed strand is two to four times slower than repair at other damage sites. These results strongly suggest that both preferential adduct formation and slow repair lead to hotspots for mutations at codons 157, 248 and 273, and that the strand bias of bulky adduct repair is primarily responsible for the strand bias of G to T transversion mutations observed in the p53 gene in human cancers.     

Solution structure of the aminofluorene-intercalated conformer of the syn [AF]-C8-dG adduct opposite a--2 deletion site in the NarI hot spot sequence context

This paper addresses structural issues related to the capacity of aminofluorene [AF] for frameshift mutations of the -2 type on C8 covalent adduct formation at the G3 site in the d(C-G1-G2-C-G3-C-C) NarI hot spot sequence. This problem has been approached from a combined NMR and relaxation matrix analysis computational structural study of the [AF]dG adduct in the d(C-G-G-C-[AF]G-C-C).d(G-G-C-C-G) sequence context at the 12/10-mer adduct level (designated [AF]dG.del(-2) 12/10-mer). The proton spectra of this system are of exceptional quality and are consistent with the formation of an AF-intercalated conformer with the modified guanine in a syn alignment displaced along with the 5'-flanking cytosine residue into the major groove. The solution structure has been determined by initially incorporating intramolecular and intermolecular proton-proton distances defined by lower and upper bound deduced from NOESY spectra as restraints in molecular mechanics computations in torsion angle space and subsequently refined through restrainted molecular dynamics calculations based on a NOE distance and intensity refinement protocol. Strikingly, the [AF]dG.del(-2) 12/10-mer duplex adopts only one of two potential AF-intercalation alignments for the [AF]dG adduct opposite the -2 deletion site in the NarI sequence context with the extrusion of the dC-[AF]dG step favored completely over extrusion of the [AF]dG-dC step at the lesion site. This polarity establishes that the structural perturbation extends 5' rather than 3' to the [AF]dG lesion site in the adduct duplex. This structure of the [AF]dG adduct opposite a -2 deletion site shows distinct differences with conclusions reported on the alignment of the related acetylaminofluorene [AAF]dG adduct opposite a -2 deletion site in the identical NarI sequence context [Milhe, C., Fuchs, R. P. P., and Lefevre, J. F. (1996) Eur. J. Biochem. 235, 120-127]. In that study, qualitative NMR data without computational analysis were employed to conclude that the extrusion at the lesion site occurs at the [AAF]dG-dC step for the AAF-intercalated conformer of the adduct duplex. The structure of the [AF]dG adduct opposite a -2 deletion site determined in our group provides molecular insights into the architecture of extended slipped mutagenic intermediates involving aromatic amine intercalation and base-displaced syn modified guanines in AF and, by analogy, AAF-induced mutagenesis in the NarI hot spot sequence context.     

Functional overlap in mismatch repair by human MSH3 and MSH6

Three human genes, hMSH2, hMSH3, and hMSH6, are homologues of the bacterial MutS gene whose products bind DNA mismatches to initiate strand-specific repair of DNA replication errors. Several studies suggest that a complex of hMSH2 x hMSH6 (hMutSalpha) functions primarily in repair of base x base mismatches or single extra bases, whereas a hMSH2 x hMSH3 complex (hMutSbeta) functions chiefly in repair of heteroduplexes containing two to four extra bases. In the present study, we compare results with a tumor cell line (HHUA) that is mutant in both hMSH3 and hMSH6 to results with derivative clones containing either wild-type hMSH3 or wild-type hMSH6, introduced by microcell-mediated transfer of chromosome 5 or 2, respectively. HHUA cells exhibit marked instability at 12 different microsatellite loci composed of repeat units of 1 to 4 base pairs. Compared to normal cells, HHUA cells have mutation rates at the HPRT locus that are elevated 500-fold for base substitutions and 2400-fold for single-base frameshifts. Extracts of HHUA cells are defective in strand-specific repair of substrates containing base x base mismatches or 1-4 extra bases. Transfer of either chromosome 5 (hMSH3) or 2 (hMSH6) into HHUA cells partially corrects instability at the microsatellite loci and also the substitution and frameshift mutator phenotypes at the HPRT locus. Extracts of these lines can repair some, but not all, heteroduplexes. The combined mutation rate and mismatch repair specificity data suggest that both hMSH3 and hMSH6 can independently participate in repair of replication errors containing base x base mismatches or 1-4 extra bases. Thus, these two gene products share redundant roles in controlling mutation rates in human cells.     

Guanine tetraplex formation by short DNA fragments containing runs of guanine and cytosine

Using CD spectroscopy, guanine tetraplex formation was studied with short DNA fragments in which cytosine residues were systematically added to runs of guanine either at the 5' or 3' ends. Potassium cations induced the G-tetraplex more easily with fragments having the guanine run at the 5' end, which is just an opposite tendency to what was reported for (G+T) oligonucleotides. However, the present (G+C) fragments simultaneously adopted other conformers that complicated the analysis. We demonstrate that repeated freezing/thawing, performed at low ionic strength, is a suitable method to exclusively stabilize the tetraplex in the (G+C) DNA fragments. In contrast to KCl, the repeated freeze/thaw cycles better stabilized the tetraplex with fragments having the guanine run on the 3' end. The tendency of guanine blocks to generate the tetraplex destabilized the d(G5).d(C5) duplex whose strands dissociated, giving rise to a stable tetraplex of (dG5) and single-stranded (dC5). In contrast to d(G3C3) and d(G5C5), repeated freezing/thawing induced the tetraplex even with the self-complementary d(C3G3) or d(C5G5); hence the latter oligonucleotides preferred the tetraplex to the apparently very stable duplex. The tetraplexes only included guanine blocks while the 5' end cytosines interfered neither with the tetraplex formation nor the tetraplex structure.     

FTIR and UV spectroscopy of parallel-stranded DNAs with mixed A*T/G*C sequences and their A*T/I*C analogues

The infrared spectra of parallel-stranded (ps) hairpin duplexes with mixed A*T/G*C composition and either isolated or sequential G*C pairs were studied in comparison with antiparallel-stranded (aps) duplexes and a corresponding set of molecules with hypoxanthine as a G base analogue lacking the exocyclic amino group. The ps duplexes showed the characteristic bands for the C2=O2 and C4=O4 stretching vibrations of thymine residues in trans-Watson-Crick A*T pairing at 1683 and 1668 cm-1. The latter band was superimposed on the stretching vibration of the free C6=O6 group of guanine. Substitution of guanine by hypoxanthine inhibited the formation of ps hairpin duplexes whatever the sequence, demonstrating that in the H-bonding between G and C the 2-NH2 group is necessary for stabilizing all of the investigated ps duplexes. This result is in agreement with a model of trans-Watson-Crick G*C base pairs with two H-bonds [N2H2(G)-N3(C) and N1H(G)-O2(C)]. However, trans-Watson-Crick A*T and G*C base pairs with two H-bonds are not isomorphous, which may explain the decreased stability of the ps, but not the aps, duplexes upon increasing the number of A*T/G*C steps. Molecular modeling studies performed on two of the ps duplexes reveal the existence of propeller twist for avoiding a clash between the N2(G) and N4(C) amino groups, and favorable stacking of sequential G*C base pairs. The optimized hairpin ps duplexes invariably incorporated G*C base pairs with two H-bonds, regardless of the initial structures adopted for the force field calculations.