The inefficient replication origin from yeast ribosomal DNA is naturally impaired in the ARS consensus sequence and in DNA unwinding

Ribosomal DNA (rDNA) replication origins of Saccharomyces cerevisiae are kn own to function inefficiently, both in the context of the tandem rDNA repea ts in the chromosome and as single copy autonomously replicating sequences (ARSs) in plasmids, Here we examined components of the rDNA ARS that might contribute to inefficient extrachromosomal replication. Like the efficient H4 ARS, the rDNA ARS requires a match to the 11 bp ARS consensus sequence ( ACS) and a broad non-conserved region that may contain multiple elements, i ncluding a DNA unwinding element (DUE), Using a single-strand-specific nucl ease hypersensitivity assay and by determining the superhelical density req uired for stable DNA unwinding, we found that the DNA of the rDNA ARS is no t as easily unwound as the H4 ARS, Unwinding of the rDNA ARS required addit ional energy, similar to the unwinding of mutations in the H4 ARS that stab ilize the double helix in the DUE region and impair replication, In vivo ex trachromosomal replication of the rDNA ARS was cold sensitive, like H4 ARS mutants that require additional energy to unwind the DUE region but unlike the easily unwound, wild-type H4 ARS, Impairment of replication function at reduced temperature suggests that the elevated energy requirement for DNA unwinding inherent in the wild-type rDNA ARS contributes to inefficient rep lication function. We also examined the essential ACS match in the rDNA ARS , which is known to be imperfect at one position, A point mutation in the e ssential ACS that corrects the imperfect match increased the efficiency of extrachromosomal replication, Our results reveal that the essential ACS ele ment and DNA unwinding in the rDNA ARS are naturally impaired, suggesting t hat inefficient function of the rDNA replication origin has a biological pu rpose.