DNA Polymerases 8 and E Are Required for Chromosomal Replication in Saccharomyces cerevisiae

Three DNA polymerases, cm, b, and E, are required for viability in Saccharomyces cerevisiae. We have investigated whether DNA polymerases e and 8 are required for DNA replication. Two temperature-sensitive mutations in the POL2 gene, encoding DNA polymerase r, have been identified by using the plasmid shuffle technique. Alkaline sucrose gradient analysis of DNA synthesis products in the mutant strains shows that no chromosomal-size DNA is formed after shift of an asynchronous culture to the nonpermissive temperature. The only DNA synthesis observed is a reduced quantity of short DNA fragments. The DNA profiles of replication intermediates from these mutants are similar to those observed with DNA synthesized in mutants deficient in DNA polymerase at under the same conditions. The finding that DNA replication stops upon shift to the nonpermissive temperature in both DNA polymerase aand DNA polymerase edeficient strains shows that both DNA polymerases are involved in elongation. By contrast, previous studies on poi3 mutants, deficient in DNA polymerase 8, suggested that there was considerable residual DNA synthesis at the nonpermissive temperature. We have reinvestigated the nature of DNA synthesis in pol3 mutants. We find that pol3 strains are defective in the synthesis of chromosomal-size DNA at the restrictive temperature after release from a hydroxyurea block. These results demonstrate that yeast DNA polymerase 8 is also required at the replication fork.

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