The Mouse Ms6-hm Hypervariable Microsatellite Forms a Hairpin and Two Unusual Tetraplexes

Abstract

The mouse Ms6-hm microsatellite consists of a tandem array of the pentamer d(CAGGG)n. This microsatellite is extremely hypervariable, showing a germ line mutation rate of 2.5%/gamete. The mechanism responsible for this instability is not known. The ability to form intrastrand structures is a conserved feature of many hypervariable sequences, and it has been suggested that the formation of such structures might account for instability by affecting DNA replication, repair, or recombination. Here we show that this microsatellite is able to form intrastrand structures as well. Under physiological conditions, the Ms6-hm microsatellite forms a hairpin as well as two different unusual intrastrand tetraplexes. The hairpin forms in the absence of monovalent cation and contains G.A, G.C, and G.G base pairs in a 1:1:1 ratio. In the presence of K+, a tetraplex is formed in which the adenines are unpaired and extrahelical, and the cytosines are involved in C.C pairs. In Na+, a tetraplex forms that contains C.C+ pairs, with the adenines being intrahelical and hydrogen-bonded to guanines. Tetraplex formation in the presence of Na+ requires both cytosines and adenines and might reflect the altered internal dimensions of this tetraplex, perhaps resulting from the ability of the C.C+ pairs to become intercalated in this sequence context. Our demonstration of the stabilization of tetraplexes by hydrogen bonding between adenines and guanines expands the hydrogen-bonding possibilities for tetraplexes and suggests that the category of sequences with tetraplex-forming potential may be larger than previously appreciated.