The autonomously replicating sequence (ARS) element ars3002 is associated 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 includes multiple near matches to the S. pombe ARS consensus sequence previously 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 multimer of an oligonucleotide containing three overlapping matches to the Maundrell ARS consensus sequence, we have detected several proteins in S. pombe crude extracts that bind to the oligonucleotide and ars3002. One of these proteins, ARS binding protein 1, was previously described (Abp1 [Y. Murakami, J. A. Huberman, and J. Hurwitz, Proc. Natl. Acad. Sci. USA 93:502-507, 1996]). In this report the isolation, characterization, and cloning of a second binding activity, designated ARS binding protein 2 (Abp2), are described. Purified Abp2 has an apparent molecular mass of 75 kDa. Footprinting analyses revealed that it binds preferentially to overlapping near matches to the Maundrell ARS consensus sequence. The gene abp2 was isolated, sequenced, and overexpressed in Escherichia coli. The DNA binding activity of overexpressed Abp2 was similar to that of native Abp2. The deduced amino acid sequence contains a region similar to a proline-rich motif (GRP) present in several proteins that bind A+T-rich DNA sequences. Replacement of amino acids 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. Disruption of the abp2 gene showed that the gene is not essential for cell viability. However, at elevated temperatures the null mutant was less viable than the wild type and exhibited changes in nuclear morphology. The null mutant entered mitosis with delayed kinetics when DNA replication was blocked with hydroxyurea, and advancement through mitosis led to the loss of cell viability and aberrant formation of septa. The null mutant was also sensitive to UV radiation, suggesting that Abp2 may play a role in regulating the cell cycle response to stress signals.