Abasic site stabilization by aromatic DNA base surrogates: High-affinity binding to a base- flipping DNA-methyltransferase*

DNA-methyltransferases catalyze the sequence-specific transfer of the methyl group of S-adenosylmethionine to target bases in genomic DNA. For gaining access to their target embedded within a double-helical structure, DNA-methyltransferases (DNA-MTases) rotate the target base out of the DNA helix. This base-flipping leads to the formation of an apparent abasic site. MTases such as cytosine-specific M HhaI and M HaeIII and also the repair enzyme uracil DNA glycosylase (UDG) insert amino acid side chains into the opened space and/or rearrange base-pairing. The adenine-specific DNA MTase M TaqI binds without amino acid insertion. This binding mode allows for a substitution of the orphaned thymine with larger DNA base surrogates without steric interference by inserted amino acid side chains. DNA containing pyrenyl, naphthyl, acenaphthyl, and biphenyl residues was tested in M TaqI binding studies. The synthesis of DNA building blocks required the formation of a C-glycosidic bond, which was established by using protected 1-chloro-2-deoxyribose as glycosyl donor and organocuprates as glycosyl acceptors. It is shown that all of the base surrogates enhanced the binding affinity to M TaqI. Incorporation of pyrene increased the binding affinity by a factor of 400. Interestingly, there is a correlation between the observed order of dissociation constants and the ability of a base surrogate to stabilize abasic sites in model duplexes. Nucleic acids are like proteins and carbohydrates subject to a multitude of biological modification reactions. In genomic DNA, the exocyclic amino groups of adenine or cytosine or the C5 of cytosine often carry methyl groups [1,2]. DNA methylation serves diverse cellular functions [3–6]. For example, a specific methylation pattern endows many bacteria with the ability to distinguish self from non-self DNA. Some bacteria use N6-methylation of adenine as a means for cell cycle control. Methyl groups in DNA of humans and other mammals play an important role in the regulation of gene expression. DNA-methylation is performed by methyltransferases which catalyze the sequence-specific transfer of the methyl group of S-adenosylmethionine to their respective target base [6,7]. For gaining access *Lecture presented at the symposium “Chemistry of nucleic acids”, as part of the 39th IUPAC Congress and 86th Conference of the Canadian Society for Chemistry: Chemistry at the Interfaces, Ottawa, Canada, 10–15 August 2003. Other Congress presentations are published in this issue, pp. 1295–1603. ‡Corresponding author: Tel.: 49 89 20937446; E-mail: [email protected] to their target embedded within a double-helical structure, DNA-methyltransferases (DNA-MTases) have evolved DNA-binding modes that lead to a local disruption of hydrogen-bonding and base-stacking interactions. Eventually, the target base is swung out of the interior of the helix, bound in an extrahelical conformation and placed in the vicinity of the catalytic machinery of the enzyme [8–10]. This so-called base-flipping has been first discovered in crystal structures of the MTase M Hha1 complexed with DNA (Fig. 1A) and was shown to be characteristic for many other DNA-modifying enzymes, including the well-studied repair enzyme UDG-glycosylase [11]. In the process of base-flipping, an apparent abasic site is formed and any base-flipping enzyme has to cope with energetic penalty introduced by the disruption of hydrogen-bonding and base-stacking interactions. It appears that based upon existing crystal structures, different mechanisms of abasic site stabilization have evolved. For example, the cytosine-specific DNA-MTase M HhaI (Fig. 1A) inserts a glutamine side chain into the space opened after enzymatic base-flipping [11]. The glutamine residue forms hydrogen bonds with the orphaned guanine. The repair enzyme uracil DNA glycosylase (UDG) employs a similar tactic by using a leucine side chain as a mechanical wedge to “push” the uracil out I. SINGH et al. © 2004 IUPAC, Pure and Applied Chemistry 76, 1563–157