MODIFIED CURVED DNA THAT COULD ALLOW LOCAL DNA UNDERWINDING AT THE NUCLEOSOMAL PSEUDODYAD FAILS TO POSITION A NUCLEOSOME IN-VIVO

In competitive in vitro reconstitution experiments synthetic DNA compo sed of tandem repeats of the repetitive sequence (A/T)(3)NN(G/C)(3)NN, specifically the 20 bp 'TG sequence' (5'-TCGGTGTTAGAGCCTGTAAC-3'), wa s reported to associate with the histone octamer with an affinity high er than that of nucleosomally derived DNA. However,at least two groups have independently shown that tandem repeats of the TG sequence do no t accommodate a stably positioned nucleosome in vivo. It was suggested that the anisotropic flexibility of the TG sequence, governed by a 10 bp sequence periodicity, is incompatible with the required underwindi ng of the DMA helix at the nucleosome pseudodyad while maintaining a b ending preference that can be accommodated in the remainder of the nuc leosome. Here we test this hypothesis directly by studying the in vivo nucleosomal structure of modified TG sequences designed to accommodat e underwinding at the pseudodyad. We show that these modifications are not sufficient to allow stable incorporation of the TG sequence repea t into a nucleosome in vivo, but do note invasion from one end of the TG heptamer of a translationally random but rotationally constrained n ucleosome. We discuss possible reasons for the absence of nucleosomes from the TG sequence in vivo.