ISOLATION, CHARACTERIZATION, AND MOLECULAR-CLONING OF A PROTEIN (ABP2) THAT BINDS TO A SCHIZOSACCHAROMYCES-POMBE ORIGIN OF REPLICATION (ARS3002)

The autonomously replicating sequence (ARS) element ars3002 is associa ted with the most active replication origin within a cluster of three closely spaced origins on chromosome III of Schizosaccharomyces pombe. A 361-bp portion of ars3002 containing detectable. ARS activity inclu des multiple near matches to the S. pombe ARS consensus sequence previ ously reported by Maundrell et al. (K. Maundrell, A. Hutchison, and S. Shall, EMBO J. 7:2203-2209, 1988). Using a gel shift assay with a mul timer of an oligonucleotide containing three overlapping matches to th e Maundrell ARS consensus sequence, we have detected several proteins In S. pombe crude extracts that bind to the oligonucleotide and ars300 2, One of these proteins, ARS binding protein 1, was previously descri bed (Abp1 [Y. Murakami, J. A. Huberman, and J. Hurwitz, Proc. Natl. Ac ad. Sci. USA 93:502-507, 1996]). In this report the isolation, charact erization, and cloning of a second binding activity, designated ARS bi nding protein 2 (Abp2), are described, Purified Abp2 has an apparent m olecular mass of 75 kDa, Footprinting analyses revealed that it binds preferentially to overlapping near matches to the Maundrell ARS consen sus sequence, The gene abp2 was isolated, sequenced, and overexpressed in Escherichia coil, The DNA binding activity of overexpressed Abp2 w as similar to that of native Abp2. The deduced amino acid sequence con tains a region similar to a proline-rich motif (GRP) present in severa l proteins that bind A+T-rich DNA sequences, Replacement of amino acid s within this motif with alanine either abolished or markedly reduced the DNA binding activity of the mutated Abp2 protein, indicating that this motif is essential for the DNA binding activity of Abp2. Disrupti on of the abp2 gene showed that the gene is not essential for cell via bility. However, at elevated temperatures the null mutant was less via ble than the wild type and exhibited changes in nuclear morphology, Th e null mutant entered mitosis with delayed kinetics when DNA replicati on was blocked with hydroxyurea, and advancement through mitosis led t o the loss of cell viability and aberrant formation of septa. The null mutant was also sensitive to UV radiation, suggesting that Abp2 may p lay a role in regulating the cell cycle response to stress signals.