Conditional inactivation of replication proteins in fission yeast using hormone-binding domains

Chen-Chun Pai; Jasmin Schnick; Stuart MacNeill; Stephen Kearsey

doi:10.1016/j.ymeth.2012.03.032

Conditional inactivation of replication proteins in fission yeast using hormone-binding domains

Chen-Chun Pai, Jasmin Schnick, Stuart MacNeill, Stephen Kearsey

Research output: Contribution to journal › Article › peer-review

Abstract

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 β-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.

Original language	English
Journal	Methods
Early online date	5 Apr 2012
DOIs	https://doi.org/10.1016/j.ymeth.2012.03.032
Publication status	Published - 2012

Keywords

β-estradiol
GINS
Hsp90
Hormone-binding domain
Schizosaccharomyces pombe
Conditional allele

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10.1016/j.ymeth.2012.03.032

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title = "Conditional inactivation of replication proteins in fission yeast using hormone-binding domains",

abstract = "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 β-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.",

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T1 - Conditional inactivation of replication proteins in fission yeast using hormone-binding domains

AU - Pai, Chen-Chun

AU - Schnick, Jasmin

AU - MacNeill, Stuart

AU - Kearsey, Stephen

PY - 2012

Y1 - 2012

N2 - 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 β-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.

AB - 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 β-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.

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KW - GINS

KW - Hsp90

KW - Hormone-binding domain

KW - Schizosaccharomyces pombe

KW - Conditional allele

UR - http://www.sciencedirect.com/science/article/pii/S1046202312000886

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DO - 10.1016/j.ymeth.2012.03.032

M3 - Article

SN - 1046-2023

JO - Methods

JF - Methods

ER -

Conditional inactivation of replication proteins in fission yeast using hormone-binding domains

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Keywords

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