Purification, gene cloning, and reconstitution of the heterotrimeric single-stranded DNA-binding protein from Schizosaccharomyces pombe.
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.
Ishiai
M
M
Graduate Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Sanchez
J P
JP
Amin
A A
AA
Murakami
Y
Y
Hurwitz
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D001667:Binding, Competitive; D002453:Cell Cycle; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics*; D005800:Genes, Fungal; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011994:Recombinant Proteins; D015183:Restriction Mapping; D012568:Schizosaccharomyces / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D046911:Macromolecular Substances; D010750:Phosphoproteins; D011994:Recombinant Proteins; C101142:SSB1 protein, S pombe; C101143:SSB2 protein, S pombe; C101144:SSB3 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins