Methylation of the EcoRI recognition site does not alter DNA conformation: the crystal structure of d(CGCGAm6ATTCGCG) at 2.0-A resolution.

Exposure of cells to alkylating agents produces DNA lesions, most of which are repaired. However some alkyl lesions persist and play a role in inducing point mutations and the subsequent carcinogenic conversion. O6-Ethylguanine (e6G) is a relatively persistent alkylation lesion caused by the exposure of DNA to N-ethyl-N-nitrosourea. We study the consequence of the e6G incorporation in DNA by X-ray crystallography. We have obtained crystals of the modified DNA dodecamer d(CGC[e6G]AATTCGCG) and the unmodified d(CGCGAATTCGCG), complexed to the minor groove binding drug netropsin. The space group of both crystals is P2(1)2(1)2(1), isomorphous to other related dodecamer DNA crystals. The structures have been solved by the molecular replacement method and refined by the constrained least-squares procedure to R-factors of approximately 16% at resolution of approximately 2.5 A. The two independent e6G-C base pairs in the DNA duplex adopt different base-pairing schemes. The e6G4-C21 base pair has a configuration similar to a normal Watson-Crick base pair, except with one three-centered hydrogen bond pair and one direct hydrogen bond between e6G4 and C21. In contrast, the e6G16-C9 base pair adopts a wobble configuration. The ethyl group is in the proximal orientation (to N7) in both base pairs. These observations enrich and support those found in the crystal structure of d(CGC[e6G]AATTCGCG), complexed to minor groove binding drugs Hoechst 33258 and Hoechst 33342 [Sriram et al. (1992) EMBO J. 11, 225-232]. We suggest that a dynamic equilibrium between these two configurations for the e6G-C base pair is likely and would present an ambiguous signal to the cellular transcription, replication, or repair mechanisms. In contrast, thymine can pair with e6G in only one way, albeit imperfect, mimicking a Watson-Crick base pair. This may be a plausible explanation of why thymine is found preferentially incorporated across the e6G during replication. In addition, we analyze the influence of the alkylation lesion on DNA and the molecular details of netropsin-DNA interaction. In the present two new netropsin complexes, the netropsin spans across five base pairs (starting halfway between C3-G22 and e6G4-C21 base pairs and ending at T8-A17 base pair) in the narrow minor groove. This is in contrast to the earlier crystal structure of netropsin complexed with another DNA dodecamer having the same AATT central core sequence, d(CGCGAATT[br5C]GCG) [Kopka et al. (1985) J. Mol. Biol. 272, 390-395]. In the latter structure, the netropsin lies between G4-br5C21 and br5C9-G16 base pairs.(ABSTRACT TRUNCATED AT 400 WORDS)

Section: Resultsmentioning

confidence: 98%

Structural consequences of a carcinogenic alkylation lesion on DNA: Effect of O6-ethylguanine [e6G] on the molecular structure of d(CGC[e6G]AATTCGCG)-netropsin complex

Sriram¹,

Marel²,

Roelen³

et al. 1992

“…Various analyses of the sequence-related "Dickerson" dodecamer (Wing et al, 1980) have shown the 5/CGA375'TCG3' segment to have unusual helical parameters. DNase digestion (Lomonossoff et al, 1981), NMR analysis (Nerdal et al, 1989;Chary et al, 1987), and X-ray crystallography (Wing et al, 1980;; Frederick et al, 1988) all suggest that the helical twist in this region varies greatly. We previously showed that inosine substitution in this region also caused aberrant melting behavior while an identical substitution in the 5'GGA3'/ 5TCC3' region did not show unusual effects (Reich & Danzitz, 1991).…”

Section: Effect Of Deoxyuridinementioning

confidence: 99%

“…Structural data exist for both the DNA substrate and methylated product, providing a good starting point for investigating the details of sequence-specific recognition. The X-ray structure of the product in which the central two adenosine residues are methylated at their N6 positions highlights the proximity of these methyl groups within the major groove and reveals minimal structural changes caused by the methylation (Frederick et al 1988).…”

mentioning

confidence: 96%

EcoRI DNA methyltransferase-DNA interactions

Reich¹,

Danzitz²

1992

We present a novel strategy with synthetic hemimethylated DNA substrates containing uracil for thymine and inosine for guanosine replacements and EcoRI DNA methyltransferase to characterize the importance of major groove hydrophobic groups to the sequence-specific modification of DNA. The bacterial Mtase uses S-adenosyl-L-methionine to methylate the double-stranded DNA site 5'GAATTC3' at the N6 position of the central adenosine of each strand. Uracil substitution in either strand at the outer thymine (5'GAATUC3') causes 2.2- and 1.7-fold improvements in specificity (kcat/KmDNA). The fact that the specificity constant for the substrate containing uracil in both strands is identical to the value expected for noninteracting substitutions suggests that no significant methyltransferase-DNA interactions are altered beyond the site of either substitution. Similar analysis of the internal thymine (5'GAAUTC3') also shows these methyl groups to make a negative contribution to specificity, although the observed nonadditivity with the doubly modified substrate clearly shows methyltransferase-DNA interactions beyond the site of substitution to be affected in this case. To further probe the effect of analogue incorporation on methyltransferase-DNA interactions beyond the site of substitution, the relatively "silent" and additive uracil changes (5'GAATUC3') were combined with inosine for guanosine substitutions (e.g., 5'IAATTC3') known to have significant negative effects on specificity. In contrast to the additivity observed with the outer thymines, these studies show significant changes in methyltransferase-DNA interactions caused by the removal of the thymine methyls. Our results implicate a complex and flexible methyltransferase-DNA interface in which subtle structural changes in the substrate are transmitted over the entire canonical site.(ABSTRACT TRUNCATED AT 250 WORDS)

“…For example, the restriction methylation modification on the N6 of adenine or C5 of cytosine prevents the host DNA from being cleaved by restriction enzymes. In those examples, it has been shown that the methylation on the base does not cause any substantial conformational changes on DNA (Frederick et al, 1988). On the other hand, the same methylation on cytosine C5 in poly(dC-dG) dramatically shifts the B-Z equilibrium toward the formation of Z-DNA (Rich et al, 1984).There are other types of modifications involving the nucleic acid backbone that have profound effects on the stability and reactivity of the nucleic acids.…”

mentioning

confidence: 99%

Effects of the O2' hydroxyl group on Z-DNA conformation: structure of Z-RNA and (araC)-[Z-DNA]

Teng¹,

Liaw²,

Marel³

et al. 1989

The left-handed Z structures of two hexamers [d(CG)r(CG)d(CG) and d(CG)(araC)d(GCG)] containing ribose and arabinose residues have been solved by X-ray diffraction analysis at 1.5-A resolution. Their conformations closely resemble that of the canonical Z-DNA. The O2' hydroxyl groups of both rC and araC residues form intramolecular hydrogen bonds with N2 of the 5' guanine residue and replace the bridging water molecules in the deep groove of Z-DNA, which stabilize the guanine in the syn conformation. The araC residue can be incorporated into the Z structure readily and facilitates B to Z transition, as supported by UV absorption spectroscopic studies. In contrast, in Z-RNA the ribose of the cytidine residue is twisted in order to form the respective hydrogen bond. The potential biological roles of the modified Z-DNA containing anticancer nucleoside araC and of Z-RNA are discussed.