Purification, gene cloning, and reconstitution of the heterotrimeric single-stranded DNA-binding protein from Schizosaccharomyces pombe Journal Article
| Authors: | Ishiai, M.; Sanchez, J. P.; Amin, A. A.; Murakami, Y.; Hurwitz, J. |
| Article Title: | Purification, gene cloning, and reconstitution of the heterotrimeric single-stranded DNA-binding protein from Schizosaccharomyces pombe |
| Abstract: | We have purified a single-stranded DNA-binding protein (SSB) from Schizosaccharomyces pombe (Sp) and have shown that it is composed of three subunits of 68, 30, and 12 kDa. The SpSSB supports T antigen-dependent unwinding of SV40 ori containing DNA, but is not functional in the SV40 in vitro replication reaction. All three genes that encode the SpSSB subunit have been isolated. The cloned cDNA of the ssb1 +, encoding the p68 subunit, contains 609 amino acids (68.3 kDa), while that of the ssb2 +, encoding the p30 subunit, contains a 279 amino acids (30.3 kDa). The genomic DNA clone of the p12 subunit gene (ssb3 +) has 2 introns and an open reading frame of 104 amino acids (11.8 kDa). Significant homology is observed among the largest and middle subunits of eukaryotic SSBs, but there is poor homology among the smallest subunits. In addition, we have reconstituted the SpSSB complex by coexpression of all three subunits in Escherichia coli. The reconstituted complex is active in single-stranded DNA binding and the T antigen-dependent unwinding of SV40 ori DNA. Finally, we observed a cell cycle-dependent phosphorylation pattern of the p30 subunit of SpSSB, which is similar to that observed for the human and Saccharomyces cerevisiae SSB. |
| Keywords: | protein phosphorylation; dna binding protein; dna-binding proteins; nonhuman; dna replication; dna recombination; cell cycle; gene expression; phosphorylation; molecular cloning; cloning, molecular; double stranded dna; amino acid sequence; molecular sequence data; sequence homology, amino acid; protein purification; saccharomyces cerevisiae; dna viruses; sequence alignment; eukaryota; nucleotide sequence; escherichia coli; recombinant proteins; phosphoproteins; base sequence; binding protein; dna primers; single stranded dna; dna, single-stranded; sequence homology; dna binding; virus t antigen; molecular weight; fungal protein; schizosaccharomyces; schizosaccharomyces pombe proteins; macromolecular substances; schizosaccharomyces pombe; open reading frame; fungal proteins; binding, competitive; genes, fungal; restriction mapping; humans; priority journal; article |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 271 |
| Issue: | 34 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 1996-08-23 |
| Start Page: | 20868 |
| End Page: | 20878 |
| Language: | English |
| DOI: | 10.1074/jbc.271.34.20868 |
| PUBMED: | 8702843 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Article -- Export Date: 22 November 2017 -- Source: Scopus |
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