The Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA), encoded by the POL30 gene, is essential for DNA replication and DNA repair processes. Twenty-one site-directed mutations were constructed in the POL30 gene, each mutation changing two adjacently located charged amino acids to alanines. Although none of the mutant strains containing these double-alanine mutations as the sole source of PCNA were temperature sensitive or cold sensitive for growth, about a third of the mutants showed sensitivity to UV light. Some of those UV-sensitive mutants had elevated spontaneous mutation rates. In addition, several mutants suppressed a cold-sensitive mutation in the CDC44 gene, which encodes the large subunit of replication factor C. A cold-sensitive mutant, which was isolated by random mutagenesis, showed a terminal phenotype at the restrictive temperature consistent with a defect in DNA replication. Several mutant PCNAs were expressed and purified from Escherichia coli, and their in vitro properties were determined. The cold-sensitive mutant (pol30-52, S115P) was a monomer, rather than a trimer, in solution. This mutant was deficient for DNA synthesis in vitro. Partial restoration of DNA polymerase ␦ holoenzyme activity was achieved at 37 C but not at 14 C by inclusion of the macromolecular crowding agent polyethylene glycol in the assay. The only other mutant (pol30-6, DD41,42AA) that showed a growth defect was partially defective for interaction with replication factor C and DNA polymerase ␦ but completely defective for interaction with DNA polymerase . Two other mutants sensitive to DNA damage showed no defect in vitro. These results indicate that the latter mutants are specifically impaired in one or more DNA repair processes whereas pol30-6 and pol30-52 mutants show their primary defects in the basic DNA replication machinery with probable associated defects in DNA repair. Therefore, DNA repair requires interactions between repair-specific protein(s) and PCNA, which are distinct from those required for DNA replication.

Embryogenesis of the free-living soil nematodeCaenorhabditis elegans produces a juvenile having about 550 cells at hatching. We have determined the lineages of 182 cells by tracing the divisions of individual cells in living embryos. An invariant pattern of cleavage divisions of the egg generates a set of stem cells. These stem cells are the founders of six stem cell lineages. Each lineage has its own clock-i.e., an autonomous rhythm of synchronous cell divisions. The rhythms are maintained in spite of extensive cellular rearrangement. The rate and the orientation of the cell divisions of the cell lineages are essentially invariant among individuals. Thus, the destiny of cells seems to depend primarily on their lineage history. The anterior position of the site of origin of the stem cells in the egg relates to the rate of the cell cycle clock, suggesting intracellular preprogramming of the uncleaved egg. We used a technique that allows normal embryogenesis, from the fertilized egg to hatching, outside the parent under a cover glass. The cell lineages reported here were analyzed from tapes of two eggs recording the complete development until the animals started moving inside the egg shell. In addition, embryogenesis was recorded and analyzed in 18 other individuals from fertilization to the 30-(10 cases), the 54-, 60-, 75-, and 87-(3 cases), and the 100-(2 cases) cell stages, respectively. We also analyzed the E-cell lineage in six more individuals to make certain that all 8 E-cells divide in going to 16. After recording was terminated, it was ascertained that, under the cover glass, all 26 animals hatched and moved normally.

We also follow the topological transitions of PCNA in the various steps of the clamp-loading pathway through both steady-state and stopped-flow fluorescence studies. We find that ATP effectively drives the clamp-loading process to completion with the formation of the closed PCNA bound to DNA, whereas ATP␥S cannot. The information derived from this work complements that obtained from previous structural and mechanistic studies and provides a more complete picture of a eukaryotic clamp-loading pathway using yeast as a paradigm.clamp loader ͉ DNA replication T he replisome is a dynamic molecular complex responsible for the duplication of a genomic DNA template at the replication fork. The central components of the replisome are the replicative DNA polymerases that copy both leading-and lagging-strand DNA (reviewed in ref. 1). Processivity of DNA replication is accomplished through a clamp protein that forms a holoenzyme complex with the replicative DNA polymerase (2). The clamp proteins identified in different organisms show a common toroidal architecture with a central channel large enough to encircle duplex DNA (3). Once loaded onto duplex DNA, clamp proteins can freely slide in a 2D space. The eukaryotic clamp proliferating cell nuclear antigen (PCNA) forms a holoenzyme complex with DNA polymerases ␦ and through specific interaction(s) between the polymerase and the PCNA ring (4-6).The task of loading PCNA onto DNA at the primer-template junction is fulfilled by replication factor C (RFC) in an ATP-driven process. Eukaryotic RFC is a heteropentameric protein complex consisting of Rfc1, -2, -3, -4, and -5 subunits. Each Rfc subunit belongs to the AAA ϩ protein family that contains characteristic ATP-binding͞hydrolysis motifs and converts the chemical energy derived from ATP binding and hydrolysis to mechanic force that is required for clamp loading (7). The RFC complex shares partial similarities to its counterpart in lower organisms like bacteriophage T4 and Escherichia coli. However, the small subunits of eukaryotic RFC are more diverged compared with those in prokaryote and archaeon, which hints for a higher complexity in both structural and functional aspects. Indeed, the yeast RFC showed an interesting departure from its prokaryotic counterparts in terms of a step-wise binding of ATP molecules modulated by its interaction with either DNA or PCNA (8). Therefore, a substantial difference is expected in the mechanistic details for clamp loading as compared with its prokaryotic counterpart. Furthermore, additional RFC-like complexes have been identified that share the Rfc2-5 subunits with RFC but carry out specialized functions in the DNA damage checkpoint and in sister chromatid cohesion (reviewed in ref. 9).Although the clamp loaders from the three model systems, including yeast, E. coli, and bacteriophage T4, all use ATP for loading the clamp onto DNA, the timing and amount of ATP binding and of its hydrolysis differ. For the E. coli system, ATP binding is sufficient for opening the ␤ clamp (10), and subseque...

The proliferating cell nuclear antigen (PCNA) acts as a processivity factor for replicative DNA polymerases and is essential for DNA replication. In vitro studies have suggested a role for PCNA in the repair synthesis step ofnucleotide excision repair, and PCNA interacts with the cyclin-dependent kinase inhibitor p21. However, because of the lack of genetic evidence, it is not clear which of the DNA repair processes are in fact affected by PCNA in vivo. Here, we describe a PCNA mutation, poi30-46, that confers ultraviolet (UV) sensitivity but has no effect on growth or cell cycle progression, and the mutant pcna interacts normally with DNA polymerase 6 and e. Genetic studies indicate that the pol30-46 mutation is specifically defective in RAD6-dependent postreplicational repair of UV damaged DNA, and this mutation impairs the error-free mode of bypass repair. These results implicate a role for PCNA as an intermediary between DNA replication and postreplicational DNA repair.Proliferating cell nuclear antigen (PCNA) acts as a processivity factor for DNA polymerases (Pol) S and s (1-5). It is a homotrimer with a subunit molecular mass of 29 kDa and is highly conserved from yeast to mammalian cells. The crystal structure of yeast PCNA shows that the trimer forms a closed ring to encircle double-stranded DNA and to interact with it via nonspecific contacts (6). PCNA is loaded onto the primer terminus by replication factor C (RF-C) in an ATP-dependent reaction (7-10), and PCNA, RF-C, and pol6 are essential for the replication of simian virus 40 DNA in a reconstituted system (11). In Saccharomyces cerevisiae, null mutations in POL30 that encodes PCNA confer recessive lethality, and a conditional lethal poi30 mutation causes growth arrest at the restrictive temperature with a terminal phenotype characteristic of DNA replication defective mutants (12, 13). In addition to an essential role in DNA replication, in vitro studies have suggested an involvement of PCNA in the repair synthesis step of nucleotide excision repair (NER) (14,15). In addition, PCNA may have a role in the coordination of cell cycle progression, DNA replication, and NER. Human p21 (Cipl,, an inhibitor of cyclin-dependent protein kinase (CDK), interacts with both CDK and PCNA. Binding of p21 to the cyclin E-CDK2 complex inhibits the kinase and prevents cells from entering S phase (16). p21 also binds directly to PCNA, and, in vitro, p21 binding differentially inhibits the action of PCNA in .While the requirement of PCNA in DNA replication is well-established, because of the lack of genetic evidence, the in vivo role of PCNA in DNA repair processes remains unknown. To determine the nature of the involvement of PCNA in DNA repair, we constructed mutations in the S. cerevisiae POL30 gene by double alanine scanning mutagenesis (13). Here, we report our studies with a highly ultraviolet (UV)-sensitive mutation, poi30-46, that indicate a role for PCNA in error-free -postreplication repair (PRR) that is dependent upon RAD6.In both prokaryotes and eukar...

Yeast exonuclease 5 is encoded by the YBR163w (DEM1) gene, and this gene has been renamed EXO5. It is distantly related to the Escherichia coli RecB exonuclease class. Exo5 is localized to the mitochondria, and EXO5 deletions or nuclease-defective EXO5 mutants invariably yield petites, amplifying either the ori3 or ori5 region of the mitochondrial genome. These petites remain unstable and undergo continuous rearrangement. The mitochondrial phenotype of exo5⌬ strains suggests an essential role for the enzyme in DNA replication and recombination. No nuclear phenotype associated with EXO5 deletions has been detected. Exo5 is a monomeric 5 exonuclease that releases dinucleotides as products. It is specific for single-stranded DNA and does not hydrolyze RNA. However, Exo5 has the capacity to slide across 5 double-stranded DNA or 5 RNA sequences and resumes cutting two nucleotides downstream of the double-stranded-to-single-stranded junction or RNAto-DNA junction, respectively.Endonucleases and exonucleases are intimately involved in all aspects of DNA metabolism in the cell. In mitochondria, several constitutive nucleases have been identified that contribute to the proper maintenance of the mitochondrial genome through replication and recombination pathways. In addition, nucleases can localize to mitochondria in response to DNA stress in order to mediate appropriate DNA repair. Among the constitutive mitochondrial nucleases in Saccharomyces cerevisiae are the Nuc1 nuclease that contributes to DNA recombination efficiency and functions in apoptosis (4, 38) and the Cce1 endonuclease that resolves recombination intermediates (29). The Din7 endonuclease is a mitochondrially located 5Ј flap endonuclease related to FEN1 (20). While deletion of the gene for either of these enzymes produced marginal mitochondrial phenotypes, more severe phenotypes were observed when combined deletions of these nuclease genes were studied or when they were combined with deletions of other genes involved in DNA recombination or repair, such as MHR1 or MSH1 (20,22,27). Recently, human Dna2 was shown to localize to both the nuclear and mitochondrial compartments and to participate in mitochondrial DNA replication and base excision repair (11,39). Its function in yeast mitochondrial DNA maintenance has not been studied in detail. Finally, the 5Ј flap endonuclease FEN1, which normally functions in primer RNA degradation during Okazaki fragment maturation in the nucleus, also localizes to the mitochondrion in response to DNA damage, participating in long-patch base excision repair (19,23).Since mitochondrial function is not essential to yeast survival, dysfunction caused by mutations of the mitochondrial genome can be readily detected as a loss of respiration function, which is scored as the inability to grow on nonfermentable carbon sources. A defect in the mitochondrial DNA polymerase ␥ MIP1 results in complete loss of the mitochondrial DNA, and the mutant fails to grow on glycerol-containing media lacking glucose (14). Such cells are designated ...