We have used DNA polymerase alpha affinity chromatography to identify factors 
involved in eukaryotic DNA replication in the yeast Saccharomyces cerevisiae. 
Two proteins that bound to the catalytic subunit of DNA polymerase alpha (Pol1 
protein) are encoded by the essential genes CDC68/SPT16 and POB3. The binding of 
both proteins was enhanced when extracts 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 
coimmunoprecipitated from whole-cell extracts with antiserum directed against 
Cdc68, and Pol1 was immunoprecipitated from whole-cell extracts with antiserum 
directed against the amino terminus of Pob3, suggesting that these proteins may 
form a complex in vivo. CDC68 also interacted genetically with POL1 and CTF4 
mutations; the maximum permissive temperature of double mutants was lower than 
for any single mutant. Overexpression of Cdc68 in a pol1 mutant strain 
dramatically decreased cell viability, consistent with the formation or 
modulation of an essential complex by these proteins in vivo. A mutation in 
CDC68/SPT16 had previously been shown 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. Rowley et 
al., Mol. Cell. Biol. 11:5718-5726, 1991), with a spectrum of phenotypes similar 
to those 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 1C 
DNA content, consistent with a possible role for Cdc68 in the prereplicative 
stage of the cell cycle. The cdc68-1 mutation caused elevated rates of 
chromosome fragment loss, a phenotype characteristic of genes whose native 
products are required for normal DNA metabolism. However, this mutation did not 
affect the rate of loss or recombination for two intact chromosomes, nor did it 
affect the retention of a low-copy-number plasmid. The previously 
uncharacterized Pob3 sequence 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, we postulate 
that polymerase alpha may interact with these proteins to gain access to its 
template or to origins of replication in vivo.