Sequence Length Dictates Repeated CAG Folding in Three-Way Junctions

Genetic instability is determined not only by exogenous factors but also by the properties of DNA itself (1, 2). Such inherent mutagenicity was established through association of fragile X syndrome, spinal and bulbar muscular atrophy, and myotonic dystrophy with abnormally long CNG (N = A, T, G, or C) repeats, and subsequently ~ 30 inherited neurological diseases have been linked with expansion of tri-and tetranucleotide repeats beyond critical thresholds (3–6). Linkage between these inherited diseases and sequence length is substantiated by its correlation with phenotypic severity and progression. Furthermore, these mutations are dynamically transmitted, as repeat tracts progressively lengthen through succeeding generations. Beyond the primary information gleaned from DNA sequence and length, secondary structure is the deeper key to this class of genetic diseases, as self-folded conformations are favored by repeated sequences (7). Stem-loop hairpins are favored by CNG repeats and are implicated in repeat expansion via replication, repair, and recombination (5). To illustrate, hairpins that form on single-stranded Okazaki fragments can disrupt coordinated synthesis on the template strands, thereby extending the nascent strand on the leading template through repeated pausing and restarting of DNA polymerase (8). These self-folded moieties can also preferentially recognize and sequester proteins involved in DNA repair, thereby disrupting normal pathways that maintain the DNA integrity (9, 10). Thus, establishing the structures of these alternative forms of DNA is necessary to understand their potential broad-scale biological function. Within double-stranded DNA, repeated sequences self-associate to produce two distinct structures (11). Slipped forms occur when strands have identical lengths of complementary repeats, and the structures are distinguished from canonical duplex DNA by enhanced sensitivity to nucleases and anomalously fast electrophoretic mobility (12). Their diverse range of structures is stable to challenging environmental changes, thereby suggesting their biological viability. Slipped intermediates form when opposing strands have different lengths, and the resulting three-way junctions are implicated in DNA expansion during replication (13). Within these structures, repeat conformation depends on base sequence, with CTG repeats favoring self-associated hairpins while CAG repeats favor open, solvent-exposed loops, and this difference may originate in the lower thermodynamic stability of CAG vs CTG repeats (7). Our studies show that tract length also dictates conformation. Toward this goal, the adenine isomer 2-aminopurine is used to develop structural and energetic models of repeated CAG sequences, which are prevalent in many inherited neurological diseases (14). Fluorescent nucleobase analogs are powerful tools for assessing DNA structure and function, and 2-aminopurine is widely used for these purposes (15, 16). Single substitutions with this fluorescent adenine analog do not alter global conformations of CAG based structures, as demonstrated by electrophoresis, spectroscopic, and energetic studies. Structural motifs are identified from their characteristic fluorescence intensities and thermal stability by utilizing the strong effect of base stacking on the fluorescence quantum yield of 2-aminopurine (17, 18). The studies described in this paper are motivated by earlier studies of (CAG)8 (19, 20). The isolated oligonucleotide behaves as the expected stem-loop hairpin, as supported from intensities in the stem region that are comparable to a duplex DNA analog, and intensities in the central loop are comparable to or exceed those of a single-stranded DNA analog. However, when this same sequence is integrated into duplex DNA, the hairpin structure is lost to yield a repeat loop that is highly and uniformly solvent-exposed. Motivated by how DNA context determines conformation in repeated DNA, we sought to understand how tract length influences secondary structure in slipped intermediate forms of DNA. Utilizing dual structural and energetic perspectives offered by 2-aminopurine substitutions, the secondary structure adopted by (CAG)15 in a three-way junction was determined. Our major finding is that this longer 15 repeat sequence retains the inherent hairpin structure of the isolated oligonucleotide when it is incorporated into the three-way junction. This behavior sharply contrasts with the open loop adopted by the analogous (CAG)8 structure, and such length dependence suggests that repeat length dictates secondary structure and potential biological functions of long repeat tracts.