Evidence that POB1, a Saccharomyces cerevisiae protein that binds to DNA polymerase alpha, acts in DNA metabolism in vivo.
Potential DNA replication accessory factors from the yeast Saccharomyces cerevisiae have previously been identified by their ability to bind to DNA polymerase alpha protein affinity matrices (J. Miles and T. Formosa, Proc. Natl. Acad. Sci. USA 89:1276-1280, 1992). We have now used genetic methods to characterize the gene encoding one of these DNA polymerase alpha-binding proteins (POB1) to determine whether it plays a role in DNA replication in vivo. We find that yeast cells lacking POB1 are viable but display a constellation of phenotypes indicating defective DNA metabolism. Populations of cells lacking POB1 accumulate abnormally high numbers of enlarged large-budded cells with a single nucleus at the neck of the bud. The average DNA content in a population of cells lacking POB1 is shifted toward the G2 value. These two phenotypes indicate that while the bulk of DNA replication is completed without POB1, mitosis is delayed. Deleting POB1 also causes elevated levels of both chromosome loss and genetic recombination, enhances the temperature sensitivity of cells with mutant DNA polymerase alpha genes, causes increased sensitivity to UV radiation in cells lacking a functional RAD9 checkpoint gene, and causes an increased probability of death in cells carrying a mutation in the MEC1 checkpoint gene. The sequence of the POB1 gene indicates that it is identical to the CTF4 (CHL15) gene identified previously in screens for mutations that diminish the fidelity of chromosome transmission. These phenotypes are consistent with defective DNA metabolism in cells lacking POB1 and strongly suggest that this DNA polymerase alpha-binding protein plays a role in accurately duplicating the genome in vivo.
Miles
J
J
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City 84132.
Formosa
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular; D004257:DNA Polymerase II / Q000378:metabolism*; D004271:DNA, Fungal / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D008540:Meiosis / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D011506:Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000528:radiation effects; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature; D014466:Ultraviolet Rays
C078338:CTF4 protein, S cerevisiae; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D004257:DNA Polymerase II