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Identification, purification, and molecular cloning of autonomously replicating sequence-binding protein 1 from
Y Murakami1, J A Huberman, J Hurwitz
1Department of Viral Oncology, Institute for Virus Research, Kyoto University, Japan.
Abstract:
Autonomously replicating sequence (ARS) elements of the fission yeast Schizosaccharomyces pombe contain multiple imperfect copies of the consensus sequence reported by Maundrell et al. [Maundrell K., Hutchison, A. & Shall, S. (1988) EMBO J. 7, 2203-2209]. When cell free extracts of S. pombe were incubated with a dimer or tetramer of an oligonucleotide containing the ARS consensus sequence, several complexes were detected using a gel mobility-shift assay. The proteins forming these complexes also bind ars3002, which is the most active origin in the ura4 region of chromosome III of S. pombe. One protein, partly responsible for the binding activity observed with crude extracts, was purified to near homogeneity. It is a 60-kDa protein and was named ARS-binding protein 1 (Abp1). Abp1 preferentially binds to multiple sites in ARS 3002 and to the DNA polymer poly[d(A.T)]. The cloning and sequence of the gene coding for Abp1 revealed that it encodes a protein of 59.8 kDa (522 amino acids). Abp1 has significant homology (25% identity, 50% similarity) to the N-terminal region (approximately 300 amino acids) of the human and mouse centromere DNA-binding protein CENP-B. Because centromeres of S. pombe contain a high density of ARS elements, Abp1 may play a role connecting DNA replication and chromosome segregation.
Insights
Researchers identified ARS-binding protein 1 (Abp1) in fission yeast, a protein crucial for DNA replication origins. Abp1 binds to autonomously replicating sequence (ARS) elements and shows homology to human centromere proteins, suggesting a role in linking replication and chromosome segregation.
Area of Science:
- Molecular Biology
- Genetics
- Yeast Biology
Background:
- Fission yeast autonomously replicating sequence (ARS) elements contain multiple imperfect consensus sequences.
- These ARS elements are critical for DNA replication initiation.
Purpose of the Study:
- To identify and characterize proteins that bind to ARS elements in Schizosaccharomyces pombe.
- To investigate the function of a novel ARS-binding protein.
Main Methods:
- Gel mobility-shift assays were used to detect protein-DNA complexes with ARS consensus sequences.
- Protein purification to near homogeneity.
- Gene cloning and sequencing of the identified ARS-binding protein.
- Homology analysis with known proteins.
Main Results:
- Several protein complexes bound to ARS consensus sequences and a specific origin (ars3002).
- A 60-kDa protein, named ARS-binding protein 1 (Abp1), was purified.
- Abp1 preferentially binds to multiple sites in ars3002 and poly[d(A.T)].
- The Abp1 gene encodes a 59.8 kDa protein with significant homology to human/mouse centromere DNA-binding protein CENP-B.
Conclusions:
- Abp1 is a novel protein that binds to ARS elements in S. pombe.
- Abp1's homology to CENP-B suggests a potential role in connecting DNA replication and chromosome segregation, especially given the high density of ARS elements in S. pombe centromeres.