Interaction of the H4 autonomously replicating sequence core consensus sequence and its 3'-flanking domain.

Abstract

Yeast autonomously replicating sequence (ARS) elements are composed of a conserved 11-base-pair (bp) core consensus sequence and a less well defined 3'-flanking region. We have investigated the relationship between the H4 ARS core consensus sequence and its 3'-flanking domain. The minimal sequences necessary and sufficient for function were determined by combining external 3' and 5' deletions to produce a nested set of ARS fragments. Sequences 5' of the core consensus were dispensable for function, but at least 66 bp of 3'-flanking domain DNA was required for full ARS function. The importance of the relative orientation of the core consensus element with respect to the 3'-flanking domain was tested by precisely inverting 14 bp of DNA including the core consensus sequence by oligonucleotide mutagenesis. This core inversion mutant was defective for all ARS function, showing that a fixed relative orientation of the core consensus and 3'-flanking domain is required for function. The 3'-flanking domain of the minimal functional H4 ARS fragment contains three sequences with a 9-of-11-bp match to the core consensus. The role of these near-match sequences was tested by directed mutagenesis. When all near-match sequences with an 8-of-11-bp match or better were simultaneously disrupted by point mutations, the resulting ARS construct retained full replication function. Therefore, multiple copies of a sequence closely related to the core consensus element are not required for H4 ARS function.