ANATOMY OF THE STIMULATIVE SEQUENCES FLANKING THE ARS CONSENSUS SEQUENCE OF CHROMOSOME-VI IN SACCHAROMYCES-CEREVISIAE

We have analyzed the relationship between autonomously replicating seq uence (ARS) structure and function for three ARS (ARS605, ARS607 and A RS609) from chromosome VI of Saccharomyces cerevisiae by systematic Xh oI-linker mutation in the ARS consensus sequence (ACS) and flanking se quences, All mutations that encroached upon the ACS destroyed ARS acti vity. DNA sequences stimulative for ARS function were identified on ei ther side of the ACS of ARS605 and only on the 3'-side of the ACS of A RS607. In ARS609, however, no such stimulative sequences were observed , Base substitutions complementary to the wild-type sequence of those stimulative regions, in ARS605 and ARS607, that did not change the Del ta G of unwinding nor affected ARS activity suggests that these region s have, at least, a function as DNA-unwinding elements (DUE), ARS605, ARS607 and ARS609 DNA are of low Delta G value and showed hypersensiti vity to single-strand-specific nuclease when inserted in negatively su percoiled plasmid. Linker mutations inhibitory for ARS activity (5L11 and 7L14) also caused significant changes in local nucleotide (nt) sen sitivity within the ACS and its adjoining regions, Complementary base substitutions, however, did not affect these changes in local nt sensi tivity, These results imply that the stimulative regions flanking the ACS are necessary to produce an optimum conformation around the ACS wh ich may be important for full ARS activity.