A DNA replication enhancer in Saccharomyces cerevisiae.

Abstract

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.Replication of eukaryotic nuclear DNA is initiated at the start of the S phase and proceeds by multiple initiations along a chromosome, generating multiple replicons. However, the regulation and the mechanism of genomic DNA replication are not understood. Saccharomyces cerevisiae provides a convenient model system for studying DNA

Asymmetric Positioning of Nucleosomes and Directional Establishment of Transcriptionally Silent Chromatin by Saccharomyces cerevisiae Silencers

“…Therefore, the direction of the Abf1p site was not important for silencing by HMR-EЉ. Along this line, it is noteworthy that the orientation of the Abf1p site in an ARS is also not important for its function as a replication origin (38).…”

Mcm2 and Mcm3, two proteins important for ARS activity, are related in structure and function.

“…Interestingly, two ARS-binding proteins, ABF 1 (Diffley and Stillman 1988), identified by its affinity to ARS1, and OBF1 (Eisenberg et al 1988), identified by its affinity to ARS121, which were first thought to be different proteins based on their affinities to apparently unrelated sequences, turned out to be the same protein with broad binding specificity (Biswas and Biswas 1990). This finding suggests that if ABF1-OBF1 plays the role of an enhancer of DNA replication (Walker et al 1990), it does so by acting at a large number of ARSs.…”

The phenotype of the minichromosome maintenance mutant mcm3 is characteristic of mutants defective in DNA replication.

“…OBF1 (19,20), identified by its affinity to ARS121, has a DNAbinding specificity similar to that of ABF1, suggesting that these two ARS-binding proteins may be the same (S. Eisenberg, personal communications). Analysis of the OBF1-binding site in ARS121 indicates that this site acts as an enhancer of DNA replication in such a way that stimulation of origin function can be provided by a synthetic OBF1-binding site independent of its orientation or distance 5' from the ARS consensus sequence (71). The role of ABF1 or OBF1 in DNA replication is unclear, since deletion of its binding sites in a number of ARSs causes only a small decrease in plasmid stability (35,59,72).…”