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.

The importance of the interdomain connector loop and of the carboxy-terminal domain of Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA) for functional interaction with DNA polymerases ␦ (Pol␦) and (Pol) was investigated by site-directed mutagenesis. Two alleles, pol30-79 (IL126,128AA) in the interdomain connector loop and pol30-90 (PK252,253AA) near the carboxy terminus, caused growth defects and elevated sensitivity to DNA-damaging agents. These two mutants also had elevated rates of spontaneous mutations. The mutator phenotype of pol30-90 was due to partially defective mismatch repair in the mutant. In vitro, the mutant PCNAs showed defects in DNA synthesis. Interestingly, the pol30-79 mutant PCNA (pcna-79) was most defective in replication with Pol␦, whereas pcna-90 was defective in replication with Pol. Protein-protein interaction studies showed that pcna-79 and pcna-90 failed to interact with Pol␦ and Pol, respectively. In addition, pcna-90 was defective in interaction with the FEN-1 endo-exonuclease (RTH1 product). A loss of interaction between pcna-79 and the smallest subunit of Pol␦, the POL32 gene product, implicates this interaction in the observed defect with the polymerase. Neither PCNA mutant showed a defect in the interaction with replication factor C or in loading by this complex. Processivity of DNA synthesis by the mutant holoenzyme containing pcna-79 was unaffected on poly(dA) ⅐ oligo(dT) but was dramatically reduced on a natural template with secondary structure. A stem-loop structure with a 20-bp stem formed a virtually complete block for the holoenzyme containing pcna-79 but posed only a minor pause site for wild-type holoenzyme, indicating a function of the POL32 gene product in allowing replication past structural blocks.

In the presence of proliferating cell nuclear antigen, yeast DNA polymerase ␦ (Pol ␦) replicated DNA at a rate of 40 -60 nt/s. When downstream double-stranded DNA was encountered, Pol ␦ paused, but most replication complexes proceeded to carry out strand-displacement synthesis at a rate of 1.5 nt/s. In the presence of the flap endonuclease FEN1 (Rad27), the complex carried out nick translation (1.7 nt/s). The Dna2 nuclease/helicase alone did not efficiently promote nick translation, nor did it affect nick translation with FEN1. Maturation in the presence of DNA ligase was studied with various downstream primers. Downstream DNA primers, RNA primers, and small 5-flaps were efficiently matured by Pol ␦ and FEN1, and Dna2 did not stimulate maturation. However, maturation of long 5-flaps to which replication protein A can bind required both DNA2 and FEN1. The maturation kinetics were optimal with a slight molar excess over DNA of Pol ␦, FEN1, and proliferating cell nuclear antigen. A large molar excess of DNA ligase substantially enhanced the rate of maturation and shortened the nick-translation patch (nucleotides excised past the RNA/DNA junction before ligation) to 4 -6 nt from 8 -12 nt with equimolar ligase. These results suggest that FEN1, but not DNA ligase, is a stable component of the maturation complex.In eukaryotic cells, Okazaki fragments are efficiently matured during elongation of DNA replication. Earlier models of this process, based on in vitro replication studies of simian virus 40 DNA, implicated the FEN1 5Ј-FLAP-exo/endonuclease and RNase H1 as the main degradative enzymes to remove the RNA moiety of the Okazaki fragment and provide an appropriate gap for filling by a DNA polymerase and sealing by DNA ligase I (for reviews, see Refs. 1 and 2). Recent studies of the mutator phenotypes of RAD27 (encoding FEN1), RNH35 (encoding RNase H1), and the double mutants suggest that the two enzymes function in separate pathways (3). Rather, genetic studies have suggested that an essential nuclease/helicase, Dna2, may be an important component of the lagging-strand replication apparatus based on several criteria, including synthetic lethality of temperature-sensitive mutations in DNA2 with a deletion mutation of the RAD27 gene (4, 5). DNA2 shows genetic interactions with DNA polymerase alpha and alphaaccessory proteins (6), and the temperature sensitivity of S. pombe DNA2 mutants is suppressed by overexpression of each of several genes playing a role in the elongation and maturation of Okazaki fragments, including those encoding FEN1, DNA ligase, and DNA polymerase ␦ (Pol ␦) 1 (7). Subsequent to the demonstration of a rather inefficient helicase activity in Dna2, a 5Ј 3 3Ј-endonuclease activity was characterized, and it is the nuclease rather than the helicase that confers the essential phenotype (8 -10). In addition, DNA2 is required for the proper maintenance of telomeres (11,12).Beyond its demonstrated function in Okazaki-fragment maturation, FEN1 plays an important role in several other DNA metabolic processe...

To address the different functions of Pol ␦ and FEN1 (Rad27) in Okazaki fragment maturation, exonucleasedeficient polymerase Pol ␦-01 and Pol ␦-5DV (corresponding to alleles pol3-01-(D321A, E323A) and pol3-5DV-(D520V), respectively) were purified and characterized in this process. In the presence of the replication clamp PCNA, both wild-type and exo ؊ Pol ␦ carried out strand displacement synthesis with similar rates; however, initiation of strand displacement synthesis was much more efficient with Pol ␦-exo ؊ . When Pol ␦-exo ؊ encountered a downstream primer, it paused with 3-5 nucleotides of the primer displaced, whereas the wild type carried out precise gap filling. Consequently, in the absence of FEN1, Pol ␦ exonuclease activity was essential for closure of simple gaps by DNA ligase. Compared with wild type, Okazaki fragment maturation with Pol ␦-exo ؊ proceeded with an increased duration of nick translation prior to ligation. Maturation was efficient in the absence of Dna2 and required Dna2 only when FEN1 activity was compromised. In agreement with these results, the proposed generation of double strand breaks in pol3-exo ؊ rad27 mutants was suppressed by the overexpression of DNA2. Further genetic studies showed that pol3-exo ؊ rad27 double mutants were sensitive to alkylation damage consistent with an in vivo defect in gap filling by exonucleasedeficient Pol ␦.Efficient and faithful maturation of Okazaki fragments during DNA replication in eukaryotes depends on a coordinated degradation of the RNA primer strand by one or more nucleases along with gap-filling DNA synthesis by a replicative DNA polymerase followed by ligation of the remaining nick. Previous models based on a combination of biochemical and genetic studies have indicated a role for the flap 5Ј-endonuclease FEN1 and the nuclease/helicase Dna2 in carrying out degradation including the removal of a displaced flap and a role for DNA polymerase ␦ (Pol ␦) 1 to carry out DNA synthesis (1). However, biochemical experiments in the accompanying paper (3) indicate that the main degradative force is provided by FEN1. The activity of Dna2 becomes crucial only in cases where strand displacement proceeds to the extent that proteins inhibitory to FEN1 can bind to the displaced 5Ј-strand (2, 3). Many DNA polymerases have an intrinsic 3Ј-5Ј exonuclease activity, which corrects polymerase errors and prevents mutations. Recently, we provided genetic evidence for the action of the 3Ј-5Ј-exonuclease of Pol ␦ in the process of Okazaki fragment maturation in vivo (4, 5). This was indicated by synthetic lethality of rad27 (FEN1) mutants with several exodeficient mutants in Pol ␦ and by a dramatic increase in duplication mutations in viable pol3-exo Ϫ rad27 double mutants. We have suggested that the 3Ј-5Ј-exonuclease could be specifically involved in preventing the excessive formation of 5Ј-flaps by strand displacement synthesis.Okazaki fragment maturation is mediated by the concerted strand displacement of Pol ␦ and degradation of the displaced strand by the nucleas...