RESUMO
During DNA replication one or both strands transiently become single stranded: first at the sites where initiation of DNA synthesis occurs (known as origins of replication) and subsequently on the lagging strands of replication forks as discontinuous Okazaki fragments are generated. We report a genome-wide analysis of single-stranded DNA (ssDNA) formation in the presence of hydroxyurea during DNA replication in wild-type and checkpoint-deficient rad53 Saccharomyces cerevisiae cells. In wild-type cells, ssDNA was first observed at a subset of replication origins and later 'migrated' bi-directionally, suggesting that ssDNA formation is associated with continuously moving replication forks. In rad53 cells, ssDNA was observed at virtually every known origin, but remained there over time, suggesting that replication forks stall. Telomeric regions seemed to be particularly sensitive to the loss of Rad53 checkpoint function. Replication origins in Schizosaccharomyces pombe were also mapped using our method.
Assuntos
DNA de Cadeia Simples/genética , Genoma Fúngico , Hidroxiureia/farmacologia , Origem de Replicação/genética , Leveduras/genética , Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2 , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , DNA de Cadeia Simples/análise , Exodesoxirribonucleases/genética , Mutação/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Proteínas Serina-Treonina Quinases/genética , Fase S/efeitos dos fármacos , Fase S/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe , Telômero/genética , Telômero/metabolismo , Leveduras/efeitos dos fármacosRESUMO
Eukaryotic chromosomal replication is a complicated process with many origins firing at different efficiencies and times during S phase. Prereplication complexes are assembled on all origins in G(1) phase, and yet only a subset of complexes is activated during S phase by DDK (for Dbf4-dependent kinase) (Cdc7-Dbf4). The yeast mcm5-bob1 (P83L) mutation bypasses DDK but results in reduced intrinsic firing efficiency at 11 endogenous origins and at origins located on minichromosomes. Origin efficiency may result from Mcm5 protein assuming an altered conformation, as predicted from the atomic structure of an archaeal MCM (for minichromosome maintenance) homologue. Similarly, an intragenic mutation in a residue predicted to interact with P83L suppresses the mcm5-bob1 bypass phenotype. We propose DDK phosphorylation of the MCM complex normally results in a single, highly active conformation of Mcm5, whereas the mcm5-bob1 mutation produces a number of conformations, only one of which is permissive for origin activation. Random adoption of these alternate states by the mcm5-bob1 protein can explain both how origin firing occurs independently of DDK and why origin efficiency is reduced. Because similar mutations in mcm2 and mcm4 cannot bypass DDK, Mcm5 protein may be a unique Mcm protein that is the final target of DDK regulation.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , Proteínas Serina-Treonina Quinases/metabolismo , Origem de Replicação , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona , Cromossomos Fúngicos/metabolismo , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/genética , Eletroforese em Gel Bidimensional , Proteínas Fúngicas/genética , Componente 4 do Complexo de Manutenção de Minicromossomo , Mutação/genética , Plasmídeos , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/genética , Relação Estrutura-AtividadeRESUMO
Temporal regulation of origin activation is widely thought to explain the pattern of early- and late-replicating domains in the Saccharomyces cerevisiae genome. Recently, single-molecule analysis of replication suggested that stochastic processes acting on origins with different probabilities of activation could generate the observed kinetics of replication without requiring an underlying temporal order. To distinguish between these possibilities, we examined a clb5Delta strain, where origin firing is largely limited to the first half of S phase, to ask whether all origins nonspecifically show decreased firing (as expected for disordered firing) or if only some origins ("late" origins) are affected. Approximately half the origins in the mutant genome show delayed replication while the remainder replicate largely on time. The delayed regions can encompass hundreds of kilobases and generally correspond to regions that replicate late in wild-type cells. Kinetic analysis of replication in wild-type cells reveals broad windows of origin firing for both early and late origins. Our results are consistent with a temporal model in which origins can show some heterogeneity in both time and probability of origin firing, but clustering of temporally like origins nevertheless yields a genome that is organized into blocks showing different replication times.
Assuntos
Cromossomos Fúngicos/genética , Ciclina B/genética , Replicação do DNA , Genoma Fúngico , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Ciclina B/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , Modelos Genéticos , Mutação , Fase S , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Eukaryotic origins of DNA replication undergo activation at various times in S-phase, allowing the genome to be duplicated in a temporally staggered fashion. In the budding yeast Saccharomyces cerevisiae, the activation times of individual origins are not intrinsic to those origins but are instead governed by surrounding sequences. Currently, there are two examples of DNA sequences that are known to advance origin activation time, centromeres and forkhead transcription factor binding sites. By combining deletion and linker scanning mutational analysis with two-dimensional gel electrophoresis to measure fork direction in the context of a two-origin plasmid, we have identified and characterized a 19- to 23-bp and a larger 584-bp DNA sequence that are capable of advancing origin activation time.