Stem-loop structures of the repetitive DNA sequences located at human centromeres

摘要

The presence of the highly conserved repetitive DNA sequences in the human centromeres argues for a special role of these sequences in their biological functions - most likely achieved by the formation of unusual structures. This prompted us to carry out quantitative one- and two-dimensional nuclear magnetic resonance (lD/2D NMR) spectroscopy to determine the structural properties of the human centromeric repeats, d(AATGG)(sub n.d)(CCATT)(sub n). The studies on centromeric DNAs reveal that the complementary sequence, d(AATGG)(sub n.d)(CCATT)(sub n), adopts the usual Watson-Crick B-DNA duplex and the pyrimidine-rich d(CCATT)(sub n) strand is essentially a random coil. However, the purine-rich d(AATGG)(sub n) strand is shown to adopt unusual stem-loop structures for repeat lengths, n=2,3,4, and 6. In addition to normal Watson-Crick A(center dot)T pairs, the stem-loop structures are stabilized by mismatch A(center dot)G and G(center dot)G pairs in the stem and G-G-A stacking in the loop. Stem-loop structures of d(AATGG)n are independently verified by gel electrophoresis and nuclease digestion studies. Thermal melting studies show that the DNA repeats, d(AATGG)(sub n), are as stable as the corresponding Watson-Crick duplex d(AATGG)(sub n.d)(CCATT)(sub n). Therefore, the sequence d(AATGG)(sub n) can, indeed, nucleate a stem-loop structure at little free-energy cost and if, during mitosis, they are located on the chromosome surface they can provide specific recognition sites for kinetochore function.