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 Schi zosaccharomyces pombe (Sp) and have shown that it is composed of three subunits of 68, 30, and 12 kDa, The SpSSB supports T antigen-dependen t unwinding of SV40 ori containing DNA, but is not functional in the S V40 in vitro replication reaction. All three genes that encode the SpS SB subunit have been isolated. The cloned cDNA of the ssb1(+), encodin g 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). Signi ficant homology is observed among tbe largest and middle subunits of e ukaryotic 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 coil, The reconstituted complex is active in single-stranded DNA binding and the T antigen-dependent u nwinding 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 She human and Saccharomyces myces cerevisiae SSB.