RESUMEN
Post-replicational telomere end processing involves both extension by telomerase and resection to produce 3'-GT-overhangs that extend beyond the complementary 5'-CA-rich strand. Resection must be carefully controlled to maintain telomere length. At short de novo telomeres generated artificially by HO endonuclease in the G2 phase, we show that dna2-defective strains are impaired in both telomere elongation and sequential 5'-CA resection. At native telomeres in dna2 mutants, GT-overhangs do clearly elongate during late S phase but are shorter than in wild type, suggesting a role for Dna2 in 5'-CA resection but also indicating significant redundancy with other nucleases. Surprisingly, elimination of Mre11 nuclease or Exo1, which are complementary to Dna2 in resection of internal double strand breaks, does not lead to further shortening of GT-overhangs in dna2 mutants. A second step in end processing involves filling in of the CA-strand to maintain appropriate telomere length. We show that Dna2 is required for normal telomeric CA-strand fill-in. Yeast dna2 mutants, like mutants in DNA ligase 1 (cdc9), accumulate low molecular weight, nascent lagging strand DNA replication intermediates at telomeres. Based on this and other results, we propose that FEN1 is not sufficient and that either Dna2 or Exo1 is required to supplement FEN1 in maturing lagging strands at telomeres. Telomeres may be among the subset of genomic locations where Dna2 helicase/nuclease is essential for the two-nuclease pathway of primer processing on lagging strands.
Asunto(s)
ADN Helicasas/genética , ADN de Hongos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Telómero/genética , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Roturas del ADN de Doble Cadena , ADN Helicasas/metabolismo , Reparación del ADN , ADN de Hongos/metabolismo , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Electroforesis en Gel de Agar , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Citometría de Flujo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación , Unión Proteica , Fase S/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Telómero/metabolismoRESUMEN
Dna2 is a dual polarity exo/endonuclease, and 5' to 3' DNA helicase involved in Okazaki Fragment Processing (OFP) and Double-Strand Break (DSB) Repair. In yeast, DNA2 is an essential gene, as expected for a DNA replication protein. Suppression of the lethality of dna2Δ mutants has been found to occur by two mechanisms: overexpression of RAD27 (scFEN1) , encoding a 5' to 3' exo/endo nuclease that processes Okazaki fragments (OFs) for ligation, or deletion of PIF1, a 5' to 3' helicase involved in mitochondrial recombination, telomerase inhibition and OFP. Mapping of a novel, spontaneously arising suppressor of dna2Δ now reveals that mutation of rad9 and double mutation of rad9 mrc1 can also suppress the lethality of dna2Δ mutants. Interaction of dna2Δ and DNA damage checkpoint mutations provides insight as to why dna2Δ is lethal but rad27Δ is not, even though evidence shows that Rad27 (ScFEN1) processes most of the Okazaki fragments, while Dna2 processes only a subset.
Asunto(s)
ADN Helicasas/genética , Genes Letales , Proteínas de Saccharomyces cerevisiae/genética , Apoptosis , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , ADN/metabolismo , Roturas del ADN de Doble Cadena , ADN Helicasas/química , ADN Helicasas/metabolismo , Reparación del ADN , Endonucleasas de ADN Solapado/genética , Endonucleasas de ADN Solapado/metabolismo , Eliminación de Gen , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Telomerasa/antagonistas & inhibidores , Telomerasa/metabolismoRESUMEN
The Mre11/Rad50/Xrs2 complex initiates IR repair by binding to the end of a double-strand break, resulting in 5' to 3' exonuclease degradation creating a single-stranded 3' overhang competent for strand invasion into the unbroken chromosome. The nuclease(s) involved are not well understood. Mre11 encodes a nuclease, but it has 3' to 5', rather than 5' to 3' activity. Furthermore, mutations that inactivate only the nuclease activity of Mre11 but not its other repair functions, mre11-D56N and mre11-H125N, are resistant to IR. This suggests that another nuclease can catalyze 5' to 3' degradation. One candidate nuclease that has not been tested to date because it is encoded by an essential gene is the Dna2 helicase/nuclease. We recently reported the ability to suppress the lethality of a dna2Delta with a pif1Delta. The dna2Delta pif1Delta mutant is IR-resistant. We have determined that dna2Delta pif1Delta mre11-D56N and dna2Delta pif1Delta mre11-H125N strains are equally as sensitive to IR as mre11Delta strains, suggesting that in the absence of Dna2, Mre11 nuclease carries out repair. The dna2Delta pif1Delta mre11-D56N triple mutant is complemented by plasmids expressing Mre11, Dna2 or dna2K1080E, a mutant with defective helicase and functional nuclease, demonstrating that the nuclease of Dna2 compensates for the absence of Mre11 nuclease in IR repair, presumably in 5' to 3' degradation at DSB ends. We further show that sgs1Delta mre11-H125N, but not sgs1Delta, is very sensitive to IR, implicating the Sgs1 helicase in the Dna2-mediated pathway.
Asunto(s)
ADN Helicasas/metabolismo , Reparación del ADN , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Regulación Fúngica de la Expresión Génica , Recombinasa Rad51/metabolismo , RecQ Helicasas/metabolismo , Recombinación Genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Alelos , Daño del ADN , Modelos Biológicos , Mutación , Plásmidos/metabolismo , Saccharomyces cerevisiae/fisiología , Rayos XRESUMEN
Most replicases are multi-subunit complexes. DNA polymerase epsilon from Saccharomyces cerevisiae is composed of four subunits: Pol2p, Dpb2p, Dpb3p, and Dpb4p. Pol2p and Dpb2p are essential. To investigate a possible role for the Dpb2p subunit in maintaining the fidelity of DNA replication, we isolated temperature-sensitive mutants in the DPB2 gene. Several of the newly isolated dpb2 alleles are strong mutators, exhibiting mutation rates equivalent to pol2 mutants defective in the 3' --> 5' proofreading exonuclease (pol2-4) or to mutants defective in mismatch repair (msh6). The dpb2 pol2-4 and dpb2 msh6 double mutants show a synergistic increase in mutation rate, indicating that the mutations arising in the dpb2 mutants are due to DNA replication errors normally corrected by mismatch repair. The dpb2 mutations decrease the affinity of Dpb2p for the Pol2p subunit as measured by two-hybrid analysis, providing a possible mechanistic explanation for the loss of high-fidelity synthesis. Our results show that DNA polymerase subunits other than those housing the DNA polymerase and 3' --> 5' exonuclease are essential in controlling the level of spontaneous mutagenesis and genetic stability in yeast cells.
Asunto(s)
ADN Polimerasa II/genética , Replicación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Sustitución de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Polimerasa II/metabolismo , Cartilla de ADN , Proteínas de Unión al ADN/metabolismo , Genotipo , Datos de Secuencia Molecular , Complejos Multienzimáticos/genética , Plásmidos , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The precise machineries required for two aspects of eukaryotic DNA replication, Okazaki fragment processing (OFP) and telomere maintenance, are poorly understood. In this work, we present evidence that Saccharomyces cerevisiae Pif1 helicase plays a wider role in DNA replication than previously appreciated and that it likely functions in conjunction with Dna2 helicase/nuclease as a component of the OFP machinery. In addition, we show that Dna2, which is known to associate with telomeres in a cell-cycle-specific manner, may be a new component of the telomere replication apparatus. Specifically, we show that deletion of PIF1 suppresses the lethality of a DNA2-null mutant. The pif1delta dna2delta strain remains methylmethane sulfonate sensitive and temperature sensitive; however, these phenotypes can be suppressed by further deletion of a subunit of pol delta, POL32. Deletion of PIF1 also suppresses the cold-sensitive lethality and hydroxyurea sensitivity of the pol32delta strain. Dna2 is thought to function by cleaving long flaps that arise during OFP due to excessive strand displacement by pol delta and/or by an as yet unidentified helicase. Thus, suppression of dna2delta can be rationalized if deletion of POL32 and/or PIF1 results in a reduction in long flaps that require Dna2 for processing. We further show that deletion of DNA2 suppresses the long-telomere phenotype and the high rate of formation of gross chromosomal rearrangements in pif1Delta mutants, suggesting a role for Dna2 in telomere elongation in the absence of Pif1.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , ADN Helicasas/metabolismo , ADN Polimerasa III/metabolismo , Replicación del ADN , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Quinasa de Punto de Control 2 , Daño del ADN , Eliminación de Gen , Genes Fúngicos/genética , Metilmetanosulfonato/farmacología , Mitocondrias/metabolismo , Modelos Biológicos , Mutación/genética , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Supresión Genética/genética , Telomerasa/antagonistas & inhibidores , TemperaturaRESUMEN
To elucidate the network that maintains high fidelity genome replication, we have introduced two conditional mutant alleles of DNA2, an essential DNA replication gene, into each of the approximately 4,700 viable yeast deletion mutants and determined the fitness of the double mutants. Fifty-six DNA2-interacting genes were identified. Clustering analysis of genomic synthetic lethality profiles of each of 43 of the DNA2-interacting genes defines a network (consisting of 322 genes and 876 interactions) whose topology provides clues as to how replication proteins coordinate regulation and repair to protect genome integrity. The results also shed new light on the functions of the query gene DNA2, which, despite many years of study, remain controversial, especially its proposed role in Okazaki fragment processing and the nature of its in vivo substrates. Because of the multifunctional nature of virtually all proteins at the replication fork, the meaning of any single genetic interaction is inherently ambiguous. The multiplexing nature of the current studies, however, combined with follow-up supporting experiments, reveals most if not all of the unique pathways requiring Dna2p. These include not only Okazaki fragment processing and DNA repair but also chromatin dynamics.