The fifth essential DNA polymerase φ inSaccharomyces cerevisiaeis localized to the nucleolus and plays an important role in synthesis of rRNA

Abstract

We report that POL5 encodes the fifth essential DNA polymerase in Saccharomyces cerevisiae. Pol5p was identified and purified from yeast cell extracts and is an aphidicolin-sensitive DNA polymerase that is stimulated by yeast proliferating cell nuclear antigen (PCNA). Thus, we named Pol5p DNA polymerase . Temperature-sensitive pol5-1ϳ-3 mutants did not arrest at G 2 ͞M at the restrictive temperature. Furthermore, the polymerase active-site mutant POL5dn gene complements the lethality of ⌬pol5. These results suggest that the polymerase activity of Pol5p is not required for the in vivo function of Pol5p. rRNA synthesis was severely inhibited at the restrictive temperature in the temperature-sensitive pol5-3 mutant cells, suggesting that an essential function of Pol5p is rRNA synthesis. Pol5p is localized exclusively to the nucleolus and binds near or at the enhancer region of rRNA-encoding DNA repeating units. DNA polymerases (Pols) play important cellular roles in DNA replication, repair, and recombination. Several distinct polymerases have been identified, purified, and characterized from prokaryotic and eukaryotic cells, and these enzymes are classified into several distinct types (1-3). The best characterized eukaryotic polymerases are polymerases (Pols) ␣, ␤, ␦, , and ␥. Recent studies also identified prokaryotic and eukaryotic polymerases that perform translesion synthesis in the presence of DNA damage (3).In Saccharomyces cerevisiae, Pol ␣, ␦, and are required for chromosomal DNA replication (4), and Pol is required for both chromosomal DNA replication and sister chromatid cohesion (5). Pol IV (␤), Rev3 (Pol ), and Rad30 (Pol ) participate in DNA repair and͞or recombination (6), and Pol␥ is the mitochondria DNA (4). The complete sequence of the yeast genome identified an ORF [YEL055C (POL5)] that encodes a polypeptide resembling B-type Pols. This study characterizes the ORF in YEL055C (POL5) and shows that it encodes the fifth essential Pol in S. cerevisiae. To investigate its function in vivo, several conditionally lethal mutants and a polymerase active-site mutant of POL5 were generated. These mutants were not defective in chromosomal DNA replication, and the polymerase active-site mutant gene fully complemented the lethality of a ⌬pol5 mutation. These results suggest that Pol5p plays an essential role in a cellular function other than chromosomal DNA replication.The nucleolus is the subnuclear compartment in which most steps in the production of ribosomes occur, including synthesis of prerRNAs, pre-RNA processing and modification, and ribosome assembly. The nucleolus also plays roles in the synthesis of ribonucleoprotein particles and pre-tRNA processing (7). Subcellular localization studies showed that Pol5p exclusively colocalizes with Nop1p, which is a component of the C ϩ D small nucleolar ribonucleoprotein-binding protein complex (8), in the nucleolus. Therefore, it is possible that Pol5p may perform an essential function in the nucleolus. This possibility is consistent with the phenotype of pol5-3 ...

Myb-binding Protein 1a (Mybbp1a) Regulates Levels and Processing of Pre-ribosomal RNA

“…In budding and bakers' yeast, the protein Pol5p was identified as the potential homolog of Mybbp1a and was shown to bind to the rRNA gene repeat (60,61). In yeast, Pol5p has been connected with an essential role in rRNA synthesis.…”

The nuclear poly(A) polymerase and Exosome cofactor Trf5 is recruited cotranscriptionally to nucleolar surveillance

“…This suggests that Pol5 is unlikely a bona fide core component of UTP-A. Pol5 was previously hypothesized to be involved in Pol I termination (Shimizu et al 2002); its presence at the vicinity of the terminator would appear compatible with such a possibility.…”

“…Early characterization of UTP subcomplexes indicated that Pol5, so named because it bears similarity to DNA polymerases (Shimizu et al 2002), is likely a substoichiometric component of UTP-A (Gallagher et al 2004;Krogan et al 2004). This was not confirmed in recent affinity purifications (Perez-Fernandez et al 2007).…”

DNA Joint Dependence of Pol X Family Polymerase Action in Nonhomologous End Joining

“…The involvement of Pol␦ and Pol⑀ in base excision repair also makes them reasonable candidates (32,33). Pol, encoded by the POL5 gene, is required for rRNA synthesis (34). Alternative approaches will be needed to gain further insight into which of these polymerases might act in yeast NHEJ.…”