Sticky DNA: Effect of the Polypurine·Polypyrimidine Sequence

Abstract

The polypurine⅐polypyrimidine sequence requirements for the formation of sticky DNA were evaluated in Escherichia coli plasmid systems to determine the potential occurrence of this conformation throughout biological systems. A mirror repeat, dinucleotide tract of (GA⅐TC) 37 , which is ubiquitous in eukaryotes, formed sticky DNA, but shorter sequences of 10 or 20 repeats were inert. (GGA⅐TCC) n inserts (where n ‫؍‬ 126, 159, and 222 bp) also formed sticky DNA. As shown previously, the control sequence (GAA⅐TTC) 150 (450 bp) readily adopted the X-shaped sticky structure; however, this structure has never been found for the nonpathogenic (GAAGGA⅐TCCTTC) 65 of the same approximate length (390 bp). A sequence that is replete with polypurine⅐polypyrimidine tracts that can form triplexes and slipped structures but lacks long repeating motifs (the 2.5-kbp intron 21 sequence from the polycystic kidney disease gene 1) was also inert. Interestingly, tracts of (GAA⅐TTC) n (where n ‫؍‬ 176 or 80) readily formed sticky DNA with (GAAGGA⅐TCCTTC) 65 cloned into the same plasmid when the pair of inserts was in the direct, but not in the indirect (inverted), orientation. The stabilities of the triple base (Watson-Crick and Hoogsteen) interactions in the DNA/DNA associated triplex region of the sticky conformations account for these observations. Our results have significant chemical and biological implications for the structure and function of this unusual DNA conformation in Friedreich's ataxia.The salient clinical and molecular biological features of Friedreich's ataxia (FRDA) 1 as well as the properties of DNA threestranded structures (triplexes) and sticky DNA were reviewed in the companion paper (1). Because this novel DNA structure was discovered (2) in the long GAA⅐TTC mutation in intron 1 of the frataxin gene, which is responsible for most cases of FRDA (3-7), we wished to determine the sequence requirements for its stabilization. Sticky DNA is a polypurine⅐polypurine⅐ polypyrimidine (R⅐R⅐Y) triplex; hence, the features must include a mirror repeat sequence. However, the role of the distribution of purines and pyrimidines on the complementary strands has not been explored. Also, because a (GAA-GGA⅐TCCTTC) 65 tract is present in the same intron, the significance of the DNA sequence on triplex and sticky DNA formation may have further relevance. At present, the function of this unusual repeating hexanucleotide tract is unclear, especially because it does not track through family pedigrees with the disease or inhibit transcription (2, 8 -10).The effect of GGA⅐TCC-interrupted triplets in long GAA⅐TTC repeat tracts was investigated (10) to determine some of the sequence requirements for sticky DNA and to evaluate further the veracity of its long GAA⅐GAA⅐TTC triplex structure. Studies were conducted on a family of seven periodically substituted inserts (all ϳ130 repeats in length) which contain 0, 4, 7, 8, 11, 20, or 50% substitution of GAA⅐TTC with GGA⅐TCC triplets. A relatively small amount of substitution (less than 11%) ...

Sticky DNA Formation in Vivo Alters the Plasmid Dimer/Monomer Ratio

“…5, bottom) from a simple folded back intramolecular triplex (Fig. 5, top left) and serves as the "glue" for the adherence of the two distant regions of the DNA (13,14). Thus, new methodologies are required for this highly unusual conformation.…”

“…Triplexes and sticky DNA at long tracts of GAA⅐TTC inhibit transcription (5, 7, 9 -12, 52, 53); also, this sequence forms sticky DNA in E. coli with the repeating hexanucleotide sequence GAAGGA⅐TCCTTC (14) that also occurs in intron 1 of the FRDA gene (4). Thus, we wished to investigate the occurrence of this non-B DNA structure in living E. coli and to evaluate its biological behaviors.…”

“…RB is converted completely to the linear monomeric form after incubation of the DNA for 10 min at 80°C in 50 mM EDTA as described earlier (8,10,13,14). Thus, the heat sensitivity of RB in the absence of divalent metal ions allows us to distinguish between RB and other possible structures.…”

“…The requirement for two blocks of long GAA⅐TTC repeats can be fulfilled by either having two tracts in one recombinant plasmid or by biological dimers of the plasmids, each parent monomer containing a single repeat tract. The intermolecular formation of sticky DNA from two independent plasmid molecules, each harboring a single long GAA⅐TTC tract, has never been observed (13,14). The novel feature of sticky DNA is the association into a triplex of two long R⅐Y tracts from distant regions of a DNA molecule; hence, upon its linearization, an X-shaped structure (8, 13) is formed with the crossover point at the repeat sequences.…”

“…Hoogsteen base-pairing stabilizes the GAA⅐GAA interactions, whereas the GAA⅐TTC pairing is Watson-Crick. Two long tracts of (GAA⅐TTC) n (where n ϭ 59 -270) in the direct repeat orientation in a single DNA molecule are required for the formation of sticky DNA (13,14); if the tracts are in the indirect repeat orientation, sticky DNA cannot be formed (13,14). The requirement for two blocks of long GAA⅐TTC repeats can be fulfilled by either having two tracts in one recombinant plasmid or by biological dimers of the plasmids, each parent monomer containing a single repeat tract.…”