THE SACCHAROMYCES-CEREVISIAE DNA-POLYMERASE ALPHA-CATALYTIC SUBUNIT INTERACTS WITH CDC68 SPT16 AND WITH POB3, A PROTEIN SIMILAR TO AN HMG1-LIKE PROTEIN/

We have used DNA polymerase alpha affinity chromatography to identify factors involved in eukaryotic DNA replication in the yeast Saccharomy ces cerevisiae. Tao proteins that bound to the catalytic subunit of DN A polymerase alpha (Pol1 protein) are encoded by the essential genes C DC68/SPT16 and POB3, The binding of both proteins was enhanced when ex tracts lacking a previously characterized polymerase binding protein, Ctf4, were used. This finding suggests that Cdc68 and Pob3 may compete with Ctf4 for binding to Pol1, Pol1 and Pob3 were coimmunoprecipitate d from whole-cell extracts with antiserum directed against Cdc68, and Pol1 was immunoprecipitated from whole-cell extracts with antiserum di rected against the amino terminus of Pob3, suggesting that these prote ins may form a complex in vivo, CDC68 also interacted genetically with POLI and CTF4 mutations; the maximum pet missive temperature of doubl e mutants mas lower than for any single mutant. Overexpression of Cdc6 8 in a pol1 mutant strain dramatically decreased cell viability, consi stent with the formation or modulation of an essential complex by thes e proteins in vivo. A mutation in CDC68/SPT16 had previously been show n to cause pleiotropic effects on the regulation of transcription (J, A. Prendergrast et al., Genetics 124:81-90, 1990); E, A, Malone et al. , Mol. Cell. Biol. 11:5710-5717, 1991; A, Ron ley ct al., Mel. Cell. B iol. 11:5718-5726, 1991), with a spectrum of phenotypes similar to tho se caused by mutations in the genes encoding histone proteins H2A and H2B (Malone et al., Mol. Cell. Biol. 11:5710-5717, 1991), We show that at the nonpermissive temperature, cdc68-1 mutants arrest as unbudded cells with a IC DNA content, consistent with a possible role for Cdc68 in the prereplicative stage of the cell cycle, The cdc68-1 mutation c aused elevated rates of chromosome fragment loss, a phenotype characte ristic of genes whose native products are required for normal DNA meta bolism. However, this mutation did not affect the rate of loss or reco mbination for two intact chromosomes, nor did it affect the retention of a low-copy-number plasmid. The previously uncharacterized Pob3 sequ ence has significant amino acid sequence similarity with an HMG1-like protein from vertebrates, Based on these results and because Cdc68 has been implicated as a regulator of chromatin structure, me postulate t hat polymerase alpha may interact with these proteins to gain access t o its template or to origins of replication in vivo.