Complementary Cytosine Research Articles

Base pair conformation-dependent excision of benzo[a]pyrene diol epoxide-guanine adducts by human nucleotide excision repair enzymes.

Human nucleotide excision repair processes carcinogen-DNA adducts at highly variable rates, even at adjacent sites along individual genes. Here, we identify conformational determinants of fast or slow repair by testing excision of N2-guanine adducts formed by benzo[a]pyrene diol epoxide (BPDE), a potent and ubiquitous mutagen that induces mainly G x C-->T x A transversions and frameshift deletions. We found that human nucleotide excision repair processes the predominant (+)-trans-BPDE-N2-dG adduct 15 times less efficiently than a standard acetylaminofluorene-C8-dG lesion in the same sequence. No difference was observed between (+)-trans- and (-)-trans-BPDE-N2-dG, but excision was enhanced about 10-fold by changing the adduct configurations to either (+)-cis- or (-)-cis-BPDE-N2-dG. Conversely, excision of (+)-cis- and (-)-cis- but not (+)-trans-BPDE-N2-dG was reduced about 10-fold when the complementary cytosine was replaced by adenine, and excision of these BPDE lesions was essentially abolished when the complementary deoxyribonucleotide was missing. Thus, a set of chemically identical BPDE adducts yielded a greater-than-100-fold range of repair rates, demonstrating that nucleotide excision repair activity is entirely dictated by local DNA conformation. In particular, this unique comparison between structurally highly defined substrates shows that fast excision of BPDE-N2-dG lesions is correlated with displacement of both the modified guanine and its partner base in the complementary strand from their normal intrahelical positions. The very slow excision of carcinogen-DNA adducts located opposite deletion sites reveals a cellular strategy that minimizes the fixation of frameshifts after mutagenic translesion synthesis.

Molecular dynamics simulations of chlorambucil/DNA adducts. A structural basis for the 5'-GNC interstrand DNA crosslink formed by nitrogen mustards.

The alkylation of DNA by chlorambucil has been studied using a computational approach. Molecular dynamics simulations were performed on the fully solvated non-covalent complex, two monoadducts and a crosslinked diadduct of chlorambucil with the d(CGG3G2CGC).-d(GCG1CCCG) duplex, in which the N7 atoms of G1, G2 and G3 are potential alkylation sites. The results provide a structural basis for the preference of nitrogen mustards to crosslink DNA duplexes at a 5'-GNC site (a 1,3 crosslink, G1-G3) rather than at a 5'-GC sites (a 1,2 crosslink, G1-G2). In the non-covalent complex simulation the drug reoriented from a non-interstrand crosslinking location to a position favorable for G1-G3 diadduct formation. It proved possible to construct a G1-G3 diadduct from a structure from the non-covalent simulation, and continue the molecular dynamics calculation without further disruption of the DNA structure. A crosslinked diadduct developed with four BII conformations on the 3' side of each alkylated guanine and of their respective complementary cytosine. In the first monoadduct simulation the starting point was the same DNA conformation used in the crosslinked diadduct simulation with alkylation at G1. In this simulation the DNA deformation was reduced, with the helix returning to a more canonical form. A second monoadduct simulation was started from a canonical DNA conformation alkylated at G3. Here, no significant motion towards a potential crosslinking conformation occurred. Collectively, the results suggest that crosslink formation is dependent upon the drug orientation prior to alkylation and the required deformation of the DNA to permit 1,3 crosslinking can largely be achieved in the non-covalent complex.

Guanosine derivatives bearing an N 2-3-imidazolepropionic acid

Synthesis of a 2′-deoxyguanosine analog tethered through the exocyclic nitrogen via a 3 carbon chain to the 4-position of an imidazole is described. The imidazole forms a hydrogen bond with the 2′-hydroxyl group of a complementary cytosine bound as a Watson-Crick base pair.

2-Aminofluorene modified DNA duplex exists in two interchangeable conformations.

One- and two-dimensional NMR shows that the carcinogen 2-aminofluorene exists in two unique, interchangeable conformations when covalently bound to a model human c-H-ras1 proto-oncogene codon 61 oligomer duplex. In one conformation the 2-aminofluorene moiety protrudes out of the major groove leaving the Watson-Crick base pairing of the cytosine and 2-aminofluorene-guanine bases intact, consistent with the ability of replicating enzymes to bypass the lesion and correctly incorporate cytosine. The second form of the modified oligomer duplex may be representative of a pre-mutagenic conformation in that the 2-aminofluorene moiety is stacked within the DNA helix, disrupting base pairing between the 2-aminofluorene-modified guanine and its complementary cytosine.

1H NMR of an oligodeoxynucleotide containing a propanodeoxyguanosine adduct positioned in a (CG)3 frameshift hotspot of Salmonella typhimurium hisD3052: Hoogsteen base-pairing at pH 5.8.

The exocyclic DNA adduct 1,N2-propano-2'-deoxyguanosine (PdG) was inserted into the oligodeoxynucleotide 5'-CGC(PdG)CGGCATG-3' and annealed to the complementary oligodeoxynucleotide 5'-CATGCCGCGCG-3'. This sequence is derived from a spontaneous revertant of the hisD3052 gene in a frameshift-sensitive tester strain of Salmonella typhimurium and is a hotspot for two-base pair deletions. The solution structure of the modified duplex was examined by 1H NMR spectroscopy. The exocyclic lesions resulted in loss of Watson-Crick base-pairing capability. Modification resulted in an approximately 24 degrees C decrease in Tm of the duplex. NMR experiments revealed pH-dependent conformational equilibria, which involved the modified base pair and its 3'-neighbor base pair. At pH 5.8, the lesion resulted in a localized perturbation of the B-form helix. PdG was rotated about the glycosyl bond from the anti to the syn conformation, thus placing the propano moiety into the major groove. This resulted in the observation of a strong NOE between the imidazole proton of PdG and the anomeric proton of the attached deoxyribose. Additional NOEs were observed between the methylene protons of the propano moiety and H5 and H6 of the 5'-neighbor cytosine. An imino proton resonance from the cytosine complementary to PdG and protonated at N3, characteristic of a Hoogsteen base pair, was observed at 15 ppm, but was broadened due to exchange with water. The amino protons of the complementary cytosine were shifted downfield from the other cytosine amino protons, characteristic of a Hoogsteen-like conformation at the site of modification. A second equilibrium involved the 3'-neighbor base pair, which alternated between Watson-Crick and Hoogsteen pairing, also via rotation of the guanosine glycosyl bond from the anti to the syn conformer. The conformational exchange of the 3'-neighbor base pair was sufficiently slow on the NMR time scale to allow simultaneous observation of resonances from the Watson-Crick and the Hoogsteen conformers.

DNA interstrand cross-links of trans-diamminedichloroplatinum(II) are preferentially formed between guanine and complementary cytosine residues.

Bases in the opposite strands of DNA cross-linked by clinically ineffective trans-diamminedichloroplatinum(II) (trans-[Pt(NH3)2Cl2]) have been identified by means of three experimental approaches. These include HPLC analysis of enzymatic digests of synthetic oligonucleotide duplexes containing the interstrand cross-link, footprinting experiments on the interstrand cross-linked oligonucleotide duplexes, and termination of the duplex transcription on trans-[Pt(NH3)2Cl2]-treated fragments of plasmid DNA. The results reveal that deoxyguanine and complementary deoxycytosine residues are preferential binding sites of trans-[Pt(NH3)2Cl2] in the interstrand adducts. The interstrand cross-linking reaction was studied by means of gel electrophoresis for the cis and trans isomers. The rate of formation of interstrand cross-links was lower for the trans isomer; however, trans-[Pt(NH3)2Cl2] formed about twice the amount of interstrand cross-links as compared with the cis isomer after 48 hr. The present results are suggested to be relevant to differences in clinical activity of the two platinum(II) isomers.

Circular dichroism anisotropy of DNA with different modifications at N7 of guanine

The complexes DNA-Ag 1+, DNA-Cu 1+, protonated DNA and DNA methylated at N7 of guanine were oriented by pumping the solutions through a multi-capillary cell in the direction of a light beam. The CD components along the DNA axis, Δε ∥, and normal to it, 2 Δε ⊥, were calculated from the CD spectra of the oriented samples by the method of Chung and Holzwarth, (1975) J. Mol. Biol. 92, 449–466. It was shown that in most cases, except that of the protonated DNA, the degree of orientation was only slightly less than that for pure DNA. This demonstrated the absence of aggregation and of appreciable denaturation. In all cases the modifications of DNA give rise to a negative component 2 Δε ⊥, whose magnitude increased as the extent of modification increased. From both the CD spectra of non-oriented samples and the absorption spectra, an inference is drawn that Ag 1+ and Cu 1+ are attached to the same site as CH 3 groups i.e., to the N7 atom of guanine. Proton transfer along the H-bond from the N1 atom of G to the N3 atom of the complementary cytosine is suggested to be a result of the modifications, although the case of H +-DNA may differ from the others. Based on the CD spectra for the anisotropic components, Δε ∥ and 2 Δε ⊥, it is proposed that ligand binding is accompanied by winding of the DNA helix.