Functional characterizations of a Saccharomyces cerevisiae DNA replication enhancer, ABF1

Date of Completion

January 1998

Keywords

Biology, Molecular|Biology, Microbiology

Degree

Ph.D.

Abstract

Abf1p is a multifunctional sequence-specific DNA binding factor in Saccharomyces cerevisiae. In this thesis, four approaches were taken towards primarily understanding the role of Abf1p as a DNA replication enhancer. ^ First, the functional significance of a major Abf1p phosphorylation was investigated. The phosphorylation site was identified, and then an appropriate mutant strain was constructed and characterized. These analyses indicated that preventing the phosphorylation event does not obviously affect Abf1p function globally, or at the ARS121 origin of replication. ^ Second, Abf1p overexpression in S. cerevisiae was explored to identify ‘active’ regions of Abf1p. The use of a sickly strain made the assay very sensitive, and two regions were identified that inhibited growth when overexpressed. Analyses described below indicated that one of these regions is involved in Abf1p's role(s) in replication. ^ Third, LexA-Abf1p fusion proteins were tested for stimulation of ARS121 derivatives harboring LexA DNA binding sites. Two Abf1p regions which appear to be capable of stimulating replication were identified. Follow-up characterizations indicated that Abf1p and the discrete replication stimulatory Abf1p regions harbor a replication stimulatory activity apparently absent from alternative transcription factors and replication enhancers. ^ Fourth, utilizing the breakpoints of the LexA(BD)-Abf1p fusion proteins as a guide, the ABF1 gene was replaced with various ABF1 deletion alleles. Alleles that provided viability were also tested for ARS121 stimulation. Taken together, the results from the fusion protein analyses and the deletion allele analyses suggest that Abf1p may be a complex replication enhancer. That is, stimulation of replication by Abf1p may occur via at least three separate regions within the protein. ^ Having identified two replication-stimulatory regions with some precision, it may now be possible to identify relevant Abf1p-interacting factors. The identification of relevant Abf1p-interacting factors is necessary for understanding the mechanisms by which eukaryotic cellular replication enhancers such as Abf1p work, and may help address whether the replication-stimulatory regions of Abf1p interact with a single factor, different factors, or multiple factors. ^

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