RESUMEN
Saccharomyces cerevisiae DNA polymerase delta (Pol delta) and DNA polymerase epsilon (Pol epsilon) are replicative DNA polymerases at the replication fork. Both enzymes are stimulated by PCNA, although to different levels. To understand why and to explore the interaction with PCNA, we compared Pol delta and Pol epsilon in physical interactions with PCNA and nucleic acids (with or without RPA), and in functional assays measuring activity and processivity. Using surface plasmon resonance technique, we show that Pol epsilon has a high affinity for DNA, but a low affinity for PCNA. In contrast, Pol delta has a low affinity for DNA and a high affinity for PCNA. The true processivity of Pol delta and Pol epsilon was measured for the first time in the presence of RPA, PCNA and RFC on single-stranded DNA. Remarkably, in the presence of PCNA, the processivity of Pol delta and Pol epsilon on RPA-coated DNA is comparable. Finally, more PCNA molecules were found on the template after it was replicated by Pol epsilon when compared to Pol delta. We conclude that Pol epsilon and Pol delta exhibit comparable processivity, but are loaded on the primer-end via different mechanisms.
Asunto(s)
ADN Polimerasa III/metabolismo , ADN Polimerasa II/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , ADN/metabolismo , Cartilla de ADN , Holoenzimas/metabolismo , Proteína de Replicación A/metabolismo , Saccharomyces cerevisiae/enzimología , Moldes GenéticosRESUMEN
Ribonucleotide reductase (RNR) is an essential enzyme that provides the cell with a balanced supply of deoxyribonucleoside triphosphates for DNA replication and repair. Mutations that affect the regulation of RNR in yeast and mammalian cells can lead to genetic abnormalities and cell death. We have expressed and purified the components of the RNR system in fission yeast, the large subunit Cdc22p, the small subunit Suc22p, and the replication inhibitor Spd1p. It was proposed (Liu, C., Powell, K. A., Mundt, K., Wu, L., Carr, A. M., and Caspari, T. (2003) Genes Dev. 17, 1130-1140) that Spd1 is an RNR inhibitor, acting by anchoring the Suc22p inside the nucleus during G1 phase. Using in vitro assays with highly purified proteins we have demonstrated that Spd1 indeed is a very efficient inhibitor of fission yeast RNR, but acting on Cdc22p. Furthermore, biosensor technique showed that Spd1p binds to the Cdc22p with a KD of 2.4 microM, whereas the affinity to Suc22p is negligible. Therefore, Spd1p inhibits fission yeast RNR activity by interacting with the Cdc22p. Similar to the situation in budding yeast, logarithmically growing fission yeast increases the dNTP pools 2-fold after 3 h of incubation in the UV mimetic 4-nitroquinoline-N-oxide. This increase is smaller than the increase observed in budding yeast but of the same order as the dNTP pool increase when synchronous Schizosaccharomyces pombe cdc10 cells are going from G1 to S-phase.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiología , Desoxirribonucleótidos/metabolismo , Ribonucleótido Reductasas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/fisiología , Schizosaccharomyces/genética , Proteínas de Ciclo Celular/genética , Clonación Molecular , Replicación del ADN , Escherichia coli/genética , Fase G1 , Cinética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Ribonucleótido Reductasas/antagonistas & inhibidores , Ribonucleótido Reductasas/genética , Schizosaccharomyces/citología , Schizosaccharomyces/enzimología , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
The structure of the multisubunit yeast DNA polymerase epsilon (Pol epsilon) was determined to 20-A resolution using cryo-EM and single-particle image analysis. A globular domain comprising the catalytic Pol2 subunit is flexibly connected to an extended structure formed by subunits Dpb2, Dpb3 and Dpb4. Consistent with the reported involvement of the latter in interaction with nucleic acids, the Dpb portion of the structure directly faces a single cleft in the Pol2 subunit that seems wide enough to accommodate double-stranded DNA. Primer-extension experiments reveal that Pol epsilon processivity requires a minimum length of primer-template duplex that corresponds to the dimensions of the extended Dpb structure. Together, these observations suggest a mechanism for interaction of Pol epsilon with DNA that might explain how the structure of the enzyme contributes to its intrinsic processivity.
Asunto(s)
ADN Polimerasa II/química , ADN Polimerasa II/ultraestructura , Saccharomyces cerevisiae/enzimología , Catálisis , Microscopía por Crioelectrón , ARN Helicasas DEAD-box , ADN Polimerasa II/metabolismo , ADN de Hongos/química , ADN de Hongos/metabolismo , ADN de Hongos/ultraestructura , Modelos Moleculares , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , ARN Helicasas/química , ARN Helicasas/metabolismo , ARN Helicasas/ultraestructura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestructuraRESUMEN
When cyclobutane pyrimidine dimers stall DNA replication by DNA polymerase (Pol) delta or epsilon, a switch occurs to allow translesion synthesis by DNA polymerase eta, followed by another switch that allows normal replication to resume. In the present study, we investigate these switches using Saccharomyces cerevisiae Pol delta, Pol epsilon and Pol eta and a series of matched and mismatched primer templates that mimic each incorporation needed to completely bypass a cis-syn thymine-thymine (TT) dimer. We report a complementary pattern of substrate use indicating that enzymatic switching involving localized translesion synthesis by Pol eta and mismatch excision and polymerization by a major replicative polymerase can account for the efficient and accurate dimer bypass known to suppress sunlight-induced mutagenesis and skin cancer.
Asunto(s)
Daño del ADN , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Disparidad de Par Base , ADN Polimerasa II/metabolismo , ADN Polimerasa III/metabolismo , Exodesoxirribonucleasas/metabolismo , Modelos Genéticos , Dímeros de Pirimidina/metabolismo , Saccharomyces cerevisiae/enzimologíaRESUMEN
We have purified wild type and exonuclease-deficient four-subunit DNA polymerase epsilon (Pol epsilon) complex from Saccharomyces cerevisiae and analyzed the fidelity of DNA synthesis by the two enzymes. Wild type Pol epsilon synthesizes DNA accurately, generating single-base substitutions and deletions at average error rates of 5' exonuclease activity is less accurate to a degree suggesting that wild type Pol epsilon proofreads at least 92% of base substitution errors and at least 99% of frameshift errors made by the polymerase. Surprisingly the base substitution fidelity of exonuclease-deficient Pol epsilon is severalfold lower than that of proofreading-deficient forms of other replicative polymerases. Moreover the spectrum of errors shows a feature not seen with other A, B, C, or X family polymerases: a high proportion of transversions resulting from T.dTTP, T.dCTP, and C.dTTP mispairs. This unique error specificity and amino acid sequence alignments suggest that the structure of the polymerase active site of Pol epsilon differs from those of other B family members. We observed both similarities and differences between the spectrum of substitutions generated by proofreading-deficient Pol epsilon in vitro and substitutions occurring in vivo in a yeast strain defective in Pol epsilon proofreading and DNA mismatch repair. We discuss the implications of these findings for the role of Pol epsilon polymerase activity in DNA replication.
Asunto(s)
ADN Polimerasa II/química , Replicación del ADN , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , Disparidad de Par Base , Secuencia de Bases , Análisis Mutacional de ADN , Reparación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Electroforesis en Gel de Poliacrilamida , Exonucleasas/metabolismo , Mutación del Sistema de Lectura , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Fenotipo , Homología de Secuencia de AminoácidoRESUMEN
DNA polymerase epsilon (Pol epsilon) from Saccharomyces cerevisiae consists of four subunits (Pol2, Dpb2, Dpb3, and Dpb4) and is essential for chromosomal DNA replication. Biochemical characterizations of Pol epsilon have been cumbersome due to protease sensitivity and the limited amounts of Pol epsilon in cells. We have developed a protocol for overexpression and purification of Pol epsilon from S. cerevisiae. The native four-subunit complex was purified to homogeneity by conventional chromatography. Pol epsilon was characterized biochemically by sedimentation velocity experiments and gel filtration experiments. The stoichiometry of the four subunits was estimated to be 1:1:1:1 from colloidal Coomassie-stained gels. Based on the sedimentation coefficient (11.9 S) and the Stokes radius (74.5 A), a molecular mass for Pol epsilon of 371 kDa was calculated, in good agreement with the calculated molecular mass of 379 kDa for a heterotetramer. Furthermore, analytical equilibrium ultracentrifugation experiments support the proposed heterotetrameric structure of Pol epsilon. Thus, both DNA polymerase delta and Pol epsilon are purified as monomeric complexes, in agreement with accumulating evidence that Pol delta and Pol epsilon are located on opposite strands of the eukaryotic replication fork.