A DNA replication enhancer in Saccharomyces cerevisiae.
We have dissected the autonomously replicating sequence ARS121 using site-directed in vitro mutagenesis. Three domains important for origin function were identified; one of these is essential and contains an 11-base-pair sequence resembling the canonical ARS core consensus; the second region, deletion of which affects the efficiency of the origin, is located 3' to the T-rich strand of the essential sequence and encompasses several elements with near matches to the ARS core consensus; the third region, containing two OBF1 DNA-binding sites and located 5' to the essential sequence, also affects the efficiency of the ARS. Here we demonstrate that a synthetic OBF1 DNA-binding site can substitute for the entire third domain in origin function. A dimer of the synthetic binding site, fused to a truncated origin containing only domains one and two, restored the origin activity to the levels of the wild-type ARS. The stimulation of origin function by the synthetic binding site was relatively orientation independent and could occur at distances as far as 1 kilobase upstream to the essential domain. Based on these results we conclude that the OBF1 DNA-binding site is an enhancer of DNA replication. We suggest that the DNA-binding site and the OBF1 protein are involved in the regulation of the activation of nuclear origins of replication in Saccharomyces cerevisiae.
Walker
S S
SS
Department of Microbiology, School of Medicine, University of Connecticut Health Center, Farmington 06032.
Francesconi
S C
SC
Eisenberg
S
S
D001483:Base Sequence; D002872:Chromosome Deletion; D004261:DNA Replication; D004271:DNA, Fungal / Q000235:genetics; D004742:Enhancer Elements, Genetic; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D004271:DNA, Fungal