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  • In studies directed toward the development of sequence speci

    2020-02-13

    In studies directed toward the development of sequence-specific major-groove binding small GZD824 [11], we desired a non-intercalating molecular scaffold that could direct attached moieties into the major groove. As an initial step toward this goal, we wished to study the DNA binding mode and affinity of a series of derivatives (1a–1f) of the cationic dye crystal violet (Fig. 1) [8], which can be readily prepared by addition of Grignard reagents derived from 4-bromo-N,N-dimethylaniline to diverse ethyl 4-N,N-dialkylaminobenzoates [9]. DNA binding ligands possessing positively charged residues have a high affinity for duplex nucleic acids due to electrostatically favourable interactions with the negatively charged phosphate backbone and electronegative atoms in the major and minor grooves [10]. Dimeric (1a–1d) and trimeric (1e) derivatives of crystal violet would thus be expected to bind tightly to duplex DNA, and varying the distances between the dye units with flexible (1b–1e) and non-flexible (1a) tethers would allow us to probe the optimal distance between charges for DNA association. Monomeric derivative 1f would allow an assessment the benefits of attaching more than one dye unit to a diamine tether. Finally, the steric bulk of the triphenylmethane moieties was expected to favour ligand binding in major groove of DNA [3].
    Results and discussion
    Conclusions We have prepared a series of monomeric (1f), dimeric (1a–1d), and trimeric (1e) derivatives of the dye crystal violet that show sub-micromolar binding affinities for duplex DNA. Salt studies performed on ligands 1c and 1e have demonstrated a strong ionic component to the DNA binding affinity of these ligands; furthermore, viscosity experiments performed on ligand 1f indicate that these molecules do not intercalate the backbone of DNA. Competition binding assays performed with 1e and known major- and minor-groove binding agents, as well as ICD bands observed during CD titrations of CT DNA with 1e, strongly suggest that these molecules preferentially associate with the major groove of DNA. Binding experiments with polynucleotides suggest a shape-selective binding of the sterically bulky dye derivative 1e to AT-rich B/B*-form DNA, which contains a wider major groove. These studies indicate that positively charged triarylmethanes are non-intercalating DNA-binding moieties that may serve to direct appended groups to the major groove, the principal site of association for regulatory proteins. Further experiments to characterize the binding (by ITC) of 1a–1f to oligonucleotides of defined sequence are underway and will be reported in due course
    Experimental part
    Acknowledgments This paper is dedicated to Professor Yoshito Kishi on the occasion of his 80th birthday. We thank the National Science Foundation (CHE-1508070) and the donors of the American Chemical Society Petroleum Research fund (53693-URI) for their generous support of our research program. BC thanks the NIH MARC program (GM008395) for support. We thank Milena Balazy, Steven Ayoub, and Lizette Aburto for initial fluorescence studies on ligands 1a-1d and the melting temperature determinations in Table 1. We thank Alejandra Fausto for assistance with fluorescence-based assays.
    Introduction Primases have an important function in DNA replication. They synthesize de novo, on single-stranded DNA (ssDNA), a primer that is then extended by DNA polymerases. In the context of the replisome, primer synthesis is repeatedly required on the lagging strand. Although DNA primases are among the most error-prone polymerases, the integrity of the newly synthesized DNA is efficiently preserved. Primase-generated RNA stretches are degraded during Okazaki fragment maturation; DNA polymerases fill the gaps, and DNA ligases seal the remaining nicks (Arezi and Kuchta, 2000, Frick and Richardson, 1999, Griep, 1995, Kuchta and Stengel, 2010). Primer synthesis can be divided into two fundamental phases. The first and probably rate-limiting step is the formation of a first phosphodiester bond between the two first nucleotides, which requires hydrolysis of the triphosphate of the elongating nucleotide, whereas the triphosphate of the initiating nucleotide becomes the 5′ end of the primer (Frick and Richardson, 2001). The second phase of primer synthesis is repeated elongation of the primer by addition of ribonucleotides at the 3′ hydroxyl group until a defined primer length is reached. This phase is very similar to the chain elongation reaction of DNA polymerases, and the presence of acidic catalytic residues suggests that the extension reaction is catalyzed by the two-metal-ion mechanism of DNA polymerases (Augustin et al., 2001, Keck et al., 2000, Steitz et al., 1994). Although the chemistry of dinucleotide synthesis and primer elongation is similar and likely involves the same catalytic residues, major questions remain, in particular how the primases are able to simultaneously bind and position the three substrates (template, initiating nucleotide, and elongating nucleotide) required for dinucleotide formation and how the primase terminates primer synthesis.