Wilhelm Guschlbauer

Structures of mismatched base pairs in DNA and their recognition by the Escherichia coli mismatch repair system.

The Escherichia coli mismatch repair system does not recognize and/or repair all mismatched base pairs with equal efficiency: whereas transition mismatches (G X T and A X C) are well repaired, the repair of some transversion mismatches (e.g. A X G or C X T) appears to depend on their position in heteroduplex DNA of phage lambda. Undecamers were synthesized and annealed to form heteroduplexes with a single base‐pair mismatch in the centre and with the five base pairs flanking each side corresponding to either repaired or unrepaired heteroduplexes of lambda DNA. Nuclear magnetic resonance (n.m.r.) studies show that a G X A mismatch gives rise to an equilibrium between fully helical and a looped‐out structure. In the unrepaired G X A mismatch duplex the latter predominates, while the helical structure is predominant in the case of repaired G X A and G X T mismatches. It appears that the E. coli mismatch repair enzymes recognize and repair intrahelical mismatched bases, but not the extrahelical bases in the looped‐out structures.

Nucleoside conformations: X. An X-ray fiber diffraction study of the gels of guanine nucleosides☆

Abstract The X-ray fiber diffraction study of four guanine nucleoside gels is reported. These nucleosides form helical structures of regularly stacked piles of tetramers in an arrangement proposed by Gellert, M., Lipsett, M. and Davies, D. R. ((1962) Proc. Natl. Acad. Sci U.S. 38, 2013). The calculated Fourier transforms are compatible with the tetrameric model. The helix characteristics of the tetrameric fibers are the following: M = 2 for Guo, M = 5 3 for 8-Br-Guo, M = 4 3 for 2′,3′-O-diacetyl-Guo, M = 5 3 for dGuo. These values correspond to a unit rotation between successive bases of 45° for Guo, 54° for 8-Br-Guo and dGuo, and about 67° for (ac)2Guo. While Guo, 8-Br-Guo and dGuo appear to form continuous head-to-tail tetramer stacks, (ac)2Guo probably has a more complicated helical symmetry, possibly involving head-to-head aligned octamers.

pH induced changes in optical activity of guanine nucleosides

Optical rotatory dispersion and circular dichroism have been used to investigate the protonation of guanosine and some of its analogues. An inversion of the principal Cotton effect and the dichroic band is observed below the acid pK. It is suggested that a conformational change from the anti form above the pK to the syn form below the pK occurs. The reasons why this change should occur only in guanosine and not in adenosine are discussed.

The structures of polyinosinic acid

SummaryPolyinosinic acid [poly(I)] was studied by a variety of techniques and together with the results in the literature the following structures are proposed.1.CD spectra of poly(I) as a function of ionic strength, temperature andpH show the existence of three main forms. At neutral pH and low ionic strength (below 0.1 M NaCl) at 3 °C or 20 °C poly(I) is a single-stranded poorly stacked helix. At 3 °C above 0.6 M NaCl poly(I) forms a multi-stranded (probably four-stranded) parallel left-handed helix. At 20 °C this structure is not completely formed. Between these extremes of ionic strength mixtures of the two forms are present which evolve differently with time, depending on ionic strength and temperature.2.The acid titration of poly(I) shows three regions as a function of ionic strength, following the same pattern as the CD spectra. The ionic strength dependence of thepK. (∼1.5) above 0.6 M NaCl suggests, but does not prove that N7 of the hypoxanthines may be implicated in the hydrogen-bonding scheme. Similarly, at high ionic strength methylation on N7 is greatly reduced in rate and extent.3.It is shown that the X-ray fiber pattern of Rich could be interpreted by a fourstranded helix withM=23/2 using a hydrogen bond between N1 and N7 between neighbouring bases.

Physicochemical properties of nucleosides 3. Gel formation by 8‐bromoguanosine

Recently, the possiblity of conformational changes in guanosine and related nucleosides has aroused much interest [l-4] . Previous work from this laboratory [3] indicated that protonation of guanine nucleosides might induce a change from the anti to the syn conformation. It was therefore of interest to investigate the properties of %bromoguanosine (BrG) which for steric reasons would preferentially assume a conformation other than anti [ 51. Since only guanosine and its nucleosides form gels, while the other natural bases which are always in the anti conformation do not show this behaviour, it was investigated, if and under which conditions BrG would form a gel. The data on the optical properties of BrG gels show that the syn conformation does not hinder the formation of a gel.

The GATATC-modification enzyme EcoRV is closely related to the GATC-recognizing methyltransferases DpnII and dam from E. coli and phage T4.

The amino acid sequence of EcoRV DNA methyltransferase which methylates the amino group of the 5′‐adenine residue of the target sequence GATATC has been found to be closely related to that of three other adenine methyltransferases, DpnII, dam and damT4, the target sequence of which is GATC. Despite large differences on the DNA level, the four sequences show four blocks of homologies. One of these blocks has the sequence DVYXDPPY and is found with little modification in numerous other DNA inethyltransferases. It is speculated that it could be the binding site of the methyl donor, S‐adenosylmethionine. On the other hand, the identification of a DNA‐binding region is more tenuous. As expected, no analogies with (dimeric) repressors and cro proteins which have the characteristic helix‐turn‐helix motif have been observed.

Nucleoside conformations: 16. Nuclear magnetic resonance and circular dichroism studies on pyrimidine-2′-fluoro-2′-deoxyribonucleosides

Abstract From the proton and fluorine NMR spectra of 3′,5′-di- O -acetyl-2′fluoro-2′-deoxyuridine, its thiouracil analogue and 2′-fluoro-2′-deoxycytidine at 250 MHz, it was concluded that these compounds are preferentially in a 3′-endo—4′-exo conformation (N-form) with only about 10–20% 2′-endo contribution (S-form). Despite the small van der Waals radius of fluorine, the 2′-fluoronu-cleosides studied in solution show the conformation characteristic for ribosides in the crystal structure. The solution data presented here are in good agreement with the crystal data of the first compound presented in the following paper by Suck, D., Saenger, W., Main, P., Germain, G. and Declercq, J.P. (1974) Biochim. Biophys. Acta 361, 257–265.

Z-DNA and other non-B-DNA structures are reversed to B-DNA by interaction with netropsin

The interaction between the B‐form specific ligands netropsin (Nt) and distamycin‐3 (Dst‐3) and DNA duplexes has been studied under conditions of salt concentration and low water activity that modify the polymer conformation into a non‐B DNA form, putatively a Z‐like form. Three polymers with strict alternating purine‐pyrimidine sequences and GC content from 100—0% have been tested: poly(dG—dC)·poly(dG—dC), poly(dA—dC)·poly(dG—dT) and poly(dA—dT)·poly(dA—dT). The titrations by Nt and Dst‐3 were followed by circular dichroism. Although specific binding of Nt to the Z‐form of poly(dG—dC)·poly(dG—dC) does not occur, Nt reverses this Z structure to the B‐type conformation; Dst‐3 is, however, totally inefficient. The presumed non‐B or Z‐like structure of poly(dA—dC)·poly(dG—dT) is reversed to the B‐form upon interaction with Nt; Dst‐3 also induces this reversal but at higher ligand ratios. The modified B‐structure of poly(dA—dT)·poly(dA—dT) in low water activity is efficiently reversed to the B‐form by interaction with both Nt and Dst‐3.

Nucleoside conformations: XIII - Circular dichroïsm of guanosine gels and the conformation of GpG and poly (G)(∘)

Summary The CD spectra of eighteen guanosine derivatives in the gel form have been measured. The amplitudes of the B1u exciton band appear to increase with sin γ (γ being the turn angle between the tetramer stacks obtained from x-ray work). On this plot the « unknowngels fit reasonably well, especially those of GpG and its 5′-5′ analogue. From a comparison of the gel CD spectrum of GpG with that of poly(G) it is concluded that the latter is a four-stranded helix with similar structural parameters as the GpG gel.

Protonated polynucleotide structures: X. Optical properties of poly(I) · poly(C) and its disproportionation complexes☆

Abstract The ORD and CD spectra and fluorescence measurements of complexes between poly(I) and poly(C) at different pH and ionic strength are reported. The evolution of optical activity and fluorescence closely follows the results obtained earlier on this system by spectrophotometric methods. These data clearly distinguish three different complexes: poly(I) · poly(C), poly(I) · poly(C) · poly(C+) and poly(I) · poly(C+). The latter is probably a reverse Hoogsteen paired helix. Comparison with published data on poly(dI) · poly(dC) suggest that the deoxypolymer may be a non-Watson-Crick helix.