Differential Targeting of Unpaired Bases within Duplex DNA by the Natural Compound Clerocidin: A Valuable Tool to Dissect DNA Secondary Structure

Matteo Nadai,Manlio Palumbo,Sara N Richter,Giorgio Palù

doi:10.1371/journal.pone.0052994

Abstract

Non-canonical DNA structures have been postulated to mediate protein-nucleic acid interactions and to function as intermediates in the generation of frame-shift mutations when errors in DNA replication occur, which result in a variety of diseases and cancers. Compounds capable of binding to non-canonical DNA conformations may thus have significant diagnostic and therapeutic potential. Clerocidin is a natural diterpenoid which has been shown to selectively react with single-stranded bases without targeting the double helix. Here we performed a comprehensive analysis on several non-canonical DNA secondary structures, namely mismatches, nicks, bulges, hairpins, with sequence variations in both the single-stranded region and the double-stranded flanking segment. By analysis of clerocidin reactivity, we were able to identify the exposed reactive residues which provided information on both the secondary structure and the accessibility of the non-paired sites. Mismatches longer than 1 base were necessary to be reached by clerocidin reactive groups, while 1-base nicks were promptly targeted by clerocidin; in hairpins, clerocidin reactivity increased with the length of the hairpin loop, while, interestingly, reactivity towards bulges reached a maximum in 3-base-long bulges and declined in longer bulges. Electrophoretic mobility shift analysis demonstrated that bulges longer than 3 bases (i.e. 5- and 7-bases) folded or stacked on the duplex region therefore being less accessible by the compound. Clerocidin thus represents a new valuable diagnostic tool to dissect DNA secondary structures.

Highlights

Nucleic acids are highly polymorphic: depending on the sequences and environmental conditions they may exist in a variety of secondary structures such as duplexes, triplexes, tetraplexes, bulges, hairpins, loops [1,2]
The symbol ¤ indicates bands that correspond to the oligonucleotide alkylated and cleaved by CL
The symbol * indicates bands that correspond to the oligonucleotide alkylated and cleaved by CL, with loss of CL

Summary

Introduction

Nucleic acids are highly polymorphic: depending on the sequences and environmental conditions they may exist in a variety of secondary structures such as duplexes, triplexes, tetraplexes, bulges, hairpins, loops [1,2]. Such non-canonical structures in nucleic acids are of general biological significance: they have been postulated to mediate protein-nucleic acid interactions, either by contacting protein residues directly or by producing a distinct tertiary structure to which the protein binds [3], and to function as intermediates in the generation of frameshift mutations when errors in DNA replication occur [4,5]. Bulged bases derived from replicative errors are considered the first step of frame-shift mutagenesis [6], resulting in a variety of diseases and cancers (e.g., myotonic dystrophy, Huntington’s disease, Friederich’s ataxia, and fragile X syndrome)

Methods

Results

Conclusion