No abstract

The investigation of the three-dimensional structure of the DNA aptamer d(G1G2T3-T4G5G6T7G8T9G10G11T12T13G14G15) which binds to and inhibits thrombin has been carried out by NMR methods. This DNA exhibits a number of long-range NOEs between residues which are not adjacent in sequence, which allowed the determination of the novel tertiary structure adopted. This DNA adopts a highly compact, highly symmetrical structure which consists of two tetrads of guanosine base pairs and three loops. The residues of the tetrads alternate anti-syn-anti-syn. This novel structural motif for DNA may also be relevant to the structure of telomere DNA.

Xeroderma pigmentosum (XP) patients fail to remove pyrimidine dimers caused by sunlight and, as a consequence, develop multiple cancers in areas exposed to light. The second most common sign, present in 20-30% of XP patients, is a set of neurological abnormalities caused by neuronal death in the central and peripheral nervous systems. Neural tissue is shielded from sunlight-induced DNA damage, so the cause of neurodegeneration in XP patients remains unexplained. In this study, we show that two major oxidative DNA lesions, 8-oxoguanine and thymine glycol, are excised from DNA in vitro by the same enzyme system responsible for removing pyrimidine dimers and other bulky DNA adducts. Our results suggest that XP neurological disease may be caused by defective repair of lesions that are produced in nerve cells by reactive oxygen species generated as by-products of an active oxidative metabolism.

Fluorescent dyes which are specific for duplex DNA have found a wide range of applications from staining gels to visualization of chromosomes. Porphyrin dyes have been found which are highly fluorescent in the presence of quadruplex but not duplex DNA. These dyes may offer a route to the specific detection of quadruplex DNA under biologically important conditions. There are three types of DNA quadruplex structures, and these may play important roles in telomere, centromere, triplet repeat, integration sites and other DNAs, and this first set of porphyrin dyes show some selectivity between the quadruplex types.

The DNA strand cleavage reaction catalyzed by endonuclease III from Escherichia coli (endo III) on the 3'-side of aldehyde abasic sites proceeds by a syn beta-elimination involving abstraction of the 2'-pro-S proton and formation of a trans alpha,beta-unsaturated aldose product; we previously reported the same stereochemical course for the reaction catalyzed by UV endonuclease V from bacteriophage T4 (UV endo V) [Mazumder, A., Gerlt, J. A., Rabow, L., Absalon, M. J., Stubbe, J., & Bolton, P. H. (1989) J. Am. Chem. Soc. 111, 8029-8030]. Since the UV endo V does not contain an 4Fe-4S center, the 4Fe-4S center present in endo III need not be assigned a unique role in the beta-elimination reaction. The beta-elimination reactions that occur under alkaline conditions (0.1 N NaOH) and in the presence of the tripeptide Lys-Trp-Lys proceed by anti beta-elimination mechanisms involving abstraction of the 2'-pro-R proton and formation of a trans alpha,beta-unsaturated aldose product. The different stereochemical outcomes of the enzymatic and nonenzymatic beta-elimination reactions support the hypothesis that the enzyme-catalyzed reactions may involve general-base-catalyzed abstraction of the 2'-pro-S proton by the internucleotidic phosphodiester leaving group.

two pathways are completely distinct and their major and Tomas Lindahl 1,5 forms in mammalian cells share no enzymes or other 1 Imperial Cancer Research Fund protein factors (Wood, 1996;). De-Clare Hall Laboratories fects in key activities of the BER process, such as AP South Mimms, Hertfordshire EN6 3LD endonuclease, DNA polymerase ␤, or the XRCC1-DNA United Kingdom ligase III heterodimer, lead to embryonic lethal pheno-2 Department of Genetics and Microbiology types in the mouse, indicating that repair of endogenous Centre Me ´dical Universitaire (CMU) DNA lesions is essential during development (Wilson 1211 Geneva 4 and Thompson, 1997). In contrast, NER defects gener-Switzerland ally are nonlethal, and mutations in any of the 7 key 3 Department of Biochemistry genes XPA to XPG can be the cause of the inherited and Division of Cancer Biology cancer-prone disease xeroderma pigmentosum in man. Department of Radiation Oncology Deamination of cytosine to uracil in DNA is counter-Emory University School of Medicine acted by BER; the repair process involves DNA polymer-Atlanta, Georgia 30322 ase ␤-catalyzed substitution of a single dCMP residue 4 Department of Chemistry in DNA to replace the excised uracil and deoxyribose Wesleyan University phosphate moieties and has been reconstituted with Middletown, Connecticut 06459 purified human factors (Kubota et al., 1996; Nicholl et al., 1997; Srivastava et al., 1998). Oxidized DNA bases such as thymine glycol (Tg) and 8-oxoguanine are be-Summary lieved to be repaired in a similar way, although the initial step is carried out by bifunctional enzymes, which can Oxidized pyrimidines in DNA are removed by a distinct both release a damaged base by DNA glycosylase activbase excision repair pathway initiated by the DNA glyity and cleave the DNA chain at the abasic site by AP cosylase-AP lyase hNth1 in human cells. We have lyase activity. Excision of various ring-saturated and reconstituted this single-residue replacement pathring-fragmented oxidized derivatives of thymine and cytosine is due to a Tg-DNA glycosylase-AP lyase activity, way with recombinant proteins, including the AP endothe human counterpart of E. coli endonuclease III or nuclease HAP1/APE, DNA polymerase ␤, and DNA li-Nth. The three-dimensional structure of the bacterial gase III-XRCC1 heterodimer. With these proteins, the enzyme has been established (Kuo et al., 1992); the nucleotide excision repair enzyme XPG serves as a homologous human enzyme hNth1 retains relevant key cofactor for the efficient function of hNth1. XPG profeatures and has been expressed in active form from a tein promotes binding of hNth1 to damaged DNA. The cloned cDNA (Aspinwall et al., 1997; Hilbert et al., 1997). stimulation of hNth1 activity is retained in XPG cata-Characteristic structural properties include a conserved lytic site mutants inactive in nucleotide excision repair. helix-hairpin-helix region that accounts for binding of The data support the model that development of Cockthe damaged pyrimidine and also contains an active ayn...

The solution-state three-dimensional structure of the DNA aptamer d(G1G2T3T4G5G6T7G8T9G10G11T12T13G14G15) which binds to and inhibits thrombin has recently been determined by NMR methods (Wang et al., 1993). This DNA adopts a highly compact, highly symmetrical structure which consists of two tetrads of guanosine base pairs and three loops. The basic features of this three-dimensional structure are preserved when the aptamer binds to thrombin. The three-dimensional structure can be used as a basis for interpreting the relative activities of modified aptamers as well as for proposing a model for the aptamer-thrombin complex. This investigation also provides a demonstration of a novel approach to medicinal chemistry in which a wide range of molecules are synthesized, a lead molecule is identified, and the structural information on the lead compound allows for rational design of additional compounds of potential therapeutic value.

There are DNA sequences which adopt the same quadruplex structural type in the presence of sodium as in the presence of sodium and potassium. There are also sequences that appear to have a requirement for the presence of potassium for the adoption of a particular quadruplex structural type. Information about the basis for these potassium effects has been obtained by examining the structures of a set of DNAs with differing numbers of loop residues and different lengths of runs of dG residues in the presence of sodium alone and in the presence of potassium and sodium. On the basis of the results, obtained primarily via solution-state NMR, it appears that very small loops favor parallel stranded quartet structures which do not require the presence of potassium. DNAs with loops of two to four residues and runs of two dG residues can form quadruplex structures of the "edge" or "chair" type in the presence of potassium but not in the presence of sodium alone. When all of the loops contain four residues, a "crossover" or "basket" type structure can be formed in the presence of sodium as well as in the presence of sodium and potassium. Structures with runs of three or four dG residues and with loops from two to four residues can form basket or crossover type structures in the absence of potassium. The presence of a purine in a loop can block both potassium binding and formation of chair type structures. Modeling of the interactions of cations with these quadruplex structures indicates that the potassium ions required for chair type structures interact with a terminal quartet and residues in the adjacent loop.