RESUMO
The fission yeast Schizosaccharomyces pombe is a useful model for analysing DNA replication as genetic methods to allow conditional inactivation of relevant proteins can provide important information about S-phase execution. A number of strategies are available to allow regulation of protein level or activity but there are disadvantages specific to each method and this may have limitations for particular proteins or experiments. We have investigated the utility of the inducible hormone-binding domain (HBD) system, which has been described in other organisms but little used in fission yeast, for the creation of conditional-lethal replication mutants. In this method, proteins are tagged with HBD and can be regulated with ß-estradiol. In this article, we describe the application of this method in fission yeast, specifically with regard to analysis of the function of GINS, an essential component of the eukaryotic replicative helicase, the CMG complex.
Assuntos
Proteínas Cromossômicas não Histona/genética , Regulação para Baixo , Proteínas Recombinantes de Fusão/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Pontos de Checagem do Ciclo Celular/genética , Núcleo Celular/metabolismo , Proteínas Cromossômicas não Histona/biossíntese , Replicação do DNA , Estradiol/farmacologia , Estrogênios/farmacologia , Expressão Gênica/efeitos dos fármacos , Fenótipo , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes de Fusão/biossíntese , Fase S/genética , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe/biossínteseRESUMO
The MCM (mini-chromosome maintenance) complex is the core of the eukaryotic replicative helicase and comprises six proteins, Mcm2-Mcm7. In humans, a variant form of the complex has Mcm2 replaced by the MCM-BP protein. Recent results suggest that a similar complex exists in fission yeast with an essential role in DNA replication and cell cycle progression. Here, we describe the purification and subunit composition of the fission yeast MCM(Mcb1) complex. Using newly generated temperature-sensitive alleles, we show that loss of MCM(Mcb1) function leads to accumulation of DNA damage, checkpoint activation and cell cycle arrest, and provide evidence for a role for MCM(Mcb1) in meiosis.