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Topic Introduction
Analysis of Recombination and Chromosome Structure during Yeast Meiosis G. Valentin Börner1,3 and Rita S. Cha2,3 1
Center for Gene Regulation in Health and Disease, Department of Biological, Geological and Environmental Sciences, Cleveland State University, 2121 Euclid Avenue, Cleveland, Ohio 44115-2214; 2North West Cancer Research Institute, School of Medical Sciences, Bangor University, Bangor LL57 2UW, United Kingdom
Meiosis is a diploid-specific differentiation program that consists of a single round of genome duplication followed by two rounds of chromosome segregation. These events result in halving of the genetic complement, which is a requirement for formation of haploid reproductive cells (i.e., spores in yeast and gametes in animals and plants). During meiosis I, homologous maternal and paternal chromosomes (homologs) pair and separate, whereas sister chromatids remain connected at the centromeres and separate during the second meiotic division. In most organisms, accurate homolog disjunction requires crossovers, which are formed as products of meiotic recombination. For the past two decades, studies of yeast meiosis have provided invaluable insights into evolutionarily conserved mechanisms of meiosis.
BIOLOGY OF YEAST MEIOSIS
Meiosis in budding yeast is an integral part of the differentiation program that results in formation of spores, a dormant cell type that provides resistance against many types of environmental stress. Yeast meiosis is induced by deprivation of nutrients, in particular nitrogen and a fermentable carbon source. This condition relieves glucose-dependent repression of Ime1, which promotes transcription of early meiosis-specific genes (Kassir et al. 1988). Despite its specialized biological function, evolutionary conservation of the meiotic program makes yeast an attractive model system (e.g., Caryl et al. 2000; Kumar et al. 2010). SPECIAL FEATURES OF MEIOSIS Chromosome Segregation
During meiosis, one round of genome duplication is followed by two rounds of chromosome segregation. During the first segregation or meiosis I (MI), homologous chromosomes separate, resulting in halving of the ploidy from diploid to haploid (Fig. 1A). The second meiotic division or meiosis II (MII) is equational, where sister chromatids separate. As a result, four haploid nuclei are generated from a single diploid precursor cell. In yeast, each nucleus becomes engulfed by a separate spore wall during sporogenesis. All four haploid spores, the products of a single meiotic event, remain contained as a tetrad within a common cell wall structure called ascus. 3
Correspondence: [email protected]; [email protected]
© 2015 Cold Spring Harbor Laboratory Press Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top077636
970
Downloaded from http://cshprotocols.cshlp.org/ at University of Liverpool Library on November 30, 2015 - Published by Cold Spring Harbor Laboratory Press
Basic Yeast Meiosis Methodology
B
A Mitotic (equational) 2N
2 x 2N
S
Dad Mom
G0
4 x 1N
0 1 Mitotic cycle G1 S
MI (reductional) MII (equational)
Tetrad
Prophase I MI MII
S 2
G2
3
4
5
6
7
8
Sporo 9 10 Time (h)
M
0 20 40 60 80 100
Time (min)
(ii)
C
Tel1/Mec1 (i)
Resected DSB break ends
SEI
dHJ
CO MI
rad50S
dmc1Δ Tel1/Mec1 (iii)
FIGURE 1. (A) Mitotic (equational) versus meiotic divisions. (S) Mitotic or meiotic replication; (MI) meiosis I; (MII) meiosis II. (B) Timeline of key meiotic events in wild-type SK1 compared to the corresponding events in vegetative cells (Sporo) Sporogenesis. (S) Mitotic or Meiotic replication; Meiotic recombination and homolog synapsis occur during prophase I. Images above the timeline are nuclear morphologies of mono-, di-, and tetra-nucleate cells, and an ascusengulfed tetrad, observable before MI, post MI, post MII, and post sporogenesis, respectively. DNA and tubulin are stained in blue and red, respectively. (C ) Key intermediates leading to crossover (CO) formation in meiotic recombination. (i) DSB: double strand break; SEI: single-end invasions; dHJ: double Holliday junctions (Schwacha and Kleckner 1997; Hunter and Kleckner 2001). (ii) Tel1 and Mec1 are activated by DSBs and regulate Spo11-activity to maintain DSB-homeostasis and ensure IH-bias (Carballo et al. 2008, 2013). (iii) Accumulation of recombination intermediates in rad50S and dmc1Δ background, respectively, trigger Tel1/Mec1-dependent meiotic arrest and/or delay (Lydall et al. 1996; Usui et al. 2001).
Meiotic S Phase
In all organisms examined to date, several stages of meiosis occupy substantially longer time intervals than the corresponding mitotic counterparts (Fig. 1B). For example, meiotic and mitotic S-phases in budding yeast last 60–80 min and 15–20 min, respectively (Williamson et al. 1983; Cha et al. 2000). Although the rate of replication fork progression and the number of replication origins used are comparable in meiotic versus mitotic replication, delayed firing of many replication origins appears to contribute to the prolongation of meiotic S-phase (Collins and Newlon 1994; Blitzblau et al. 2012). Meiotic Recombination
In most organisms, meiotic recombination is initiated after S-phase by Spo11 catalysis of DNA double-strand breaks (DSBs) (Bergerat et al. 1997; Keeney et al. 1997). Recombinational repair of meiotic DSBs proceeds with an interhomolog (IH) bias, whereby the majority of breaks are repaired using an intact nonsister homologous chromosome as a template. IH-bias ensures formation of crossovers (COs) essential for homolog segregation (e.g., Schwacha and Kleckner 1997) (Fig. 1C). Meiotic Chromosome Structure
Meiotic recombination takes place in the context of a tripartite proteinaceous structure, called the synaptonemal complex (SC), which intimately juxtaposes homologous chromosomes (Sym et al. 1993). The extent of SC development defines substages of meiotic prophase, where leptotene, Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top077636
971
Downloaded from http://cshprotocols.cshlp.org/ at University of Liverpool Library on November 30, 2015 - Published by Cold Spring Harbor Laboratory Press
G.V. Börner and R.S. Cha
zygotene, pachytene, and diplotene refer, respectively, to stages of minimal, partial, and full synapsis as well as the stage at which the SC has disassembled.
Regulation of Meiotic Progression
Meiotic progression is regulated by factors known to also control the mitotic cell cycle (e.g., cyclindependent kinase [Cdc28] and polo kinase [Cdc5]) (e.g., Wan et al. 2008; Sourirajan and Lichten 2008). In addition, meiosis-specific proteins such as the transcription factor Ndt80 regulate meiotic progression (Xu et al. 1995). Initiation of meiotic recombination activates the conserved ATM/ATR kinases (i.e., Tel1/Mec1 in budding yeast) (Fig. 1C). During unperturbed meiosis, Tel1/Mec1 function to control the extent of Spo11-catalysis and to promote IH-bias (Carballo et al. 2008, 2013). Tel1/ Mec1 also function as bona fide checkpoint regulators, triggering meiotic arrest in response to meiotic recombination defects (Fig. 1C) (Lydall et al. 1996; Usui et al. 2001).
ANALYSIS OF MEIOSIS IN YEAST
In accompanying protocols, we present selected methods for analyzing meiotic products in budding yeast following sporulation on solid medium (Protocol: Analysis of Yeast Sporulation Efficiency, Spore Viability, and Meiotic Recombination on Solid Medium [Börner and Cha 2015a]), preparing highly synchronous meiotic cultures in liquid medium (Protocol: Induction and Analysis of Synchronous Meiotic Yeast Cultures [Börner and Cha 2015b]), and analyzing meiotic recombination and chromosome dynamics in samples collected from synchronous meiotic cultures (Protocol: Analysis of Meiotic Recombination and Homolog Interaction during Yeast Meiosis [Börner and Cha 2015c]). These protocols provide a practical introduction for the novice to the study of yeast meiosis. Additional yeast meiosis methods (e.g., screening for and isolating meiotic mutants) as well as methods for investigating meiosis in other organisms have been published elsewhere (Guthrie and Fink 1991; Keeney 2009a,b).
Choice of Strain Background
Several strain backgrounds have been used for meiotic analysis (e.g., Cotton et al. 2009; Elrod et al. 2009). However, the methods described in the above-mentioned protocols are optimized for SK1, which is a readily sporulating strain of Saccharomyces cerevisiae. The SK1 strain completes both meiotic divisions by 10 h after transfer to sporulation medium (SPM) at 30˚C (Padmore et al. 1991). Spore formation is observed in >90% of cells within 24 h and the spores typically show >90% viability (e.g., Padmore et al. 1991).
Synchronous Meiotic Cultures
Induction of synchronous meiosis entails a presporulation growth protocol that generates a large number of G0-arrested cells, which are subsequently transferred to liquid SPM (see Protocol: Induction and Analysis of Synchronous Meiotic Yeast Cultures [Börner and Cha 2015b]). In a typical synchronous culture at 30˚C, 50% of active cells have entered meiotic S-phase by 2.5 h, followed by completion of S-phase 1 h later (Padmore et al. 1991; Cha et al. 2000). Meiotic recombination is initiated after S-phase, with 50% of DSBs formed at 4 h and exit from this stage at 5 h. The 50% entry time for appearance of COs and NCOs is 6 h, and the first meiotic division ensues at 7 h (Fig. 1B) (Padmore et al. 1991). Some optimization may be required to obtain a highly synchronous SK1 meiotic culture, with variability attributable to factors such as water quality, temperature, pH, aeration, and pre-SPM growth conditions (e.g., Cha et al. 2000; Börner et al. 2004). 972
Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top077636
Downloaded from http://cshprotocols.cshlp.org/ at University of Liverpool Library on November 30, 2015 - Published by Cold Spring Harbor Laboratory Press
Basic Yeast Meiosis Methodology
Spore Formation and Spore Viability
Most mutations that affect meiosis result in either reduced spore formation and/or reduced spore viability. Reduced sporulation efficiency (see Protocol: Analysis of Yeast Sporulation Efficiency, Spore Viability, and Meiotic Recombination on Solid Medium [Börner and Cha 2015a]) may result from defects in positive drivers of meiotic progression, for example, Ndt80 or Cdc5 (see above). Alternatively, failure to complete meiosis could be due to a Tel1/Mec1-mediated recombination checkpoint response. In the latter case, introduction of mutations that prevent initiation of meiotic recombination (e.g., spo11) or abolish checkpoint response (e.g., MEC1-activating protein Rad24) restores sporulation efficiency (Lydall et al. 1996). Effects of mutations on spore viability can be substantial (
Topic Introduction
Analysis of Recombination and Chromosome Structure during Yeast Meiosis G. Valentin Börner1,3 and Rita S. Cha2,3 1
Center for Gene Regulation in Health and Disease, Department of Biological, Geological and Environmental Sciences, Cleveland State University, 2121 Euclid Avenue, Cleveland, Ohio 44115-2214; 2North West Cancer Research Institute, School of Medical Sciences, Bangor University, Bangor LL57 2UW, United Kingdom
Meiosis is a diploid-specific differentiation program that consists of a single round of genome duplication followed by two rounds of chromosome segregation. These events result in halving of the genetic complement, which is a requirement for formation of haploid reproductive cells (i.e., spores in yeast and gametes in animals and plants). During meiosis I, homologous maternal and paternal chromosomes (homologs) pair and separate, whereas sister chromatids remain connected at the centromeres and separate during the second meiotic division. In most organisms, accurate homolog disjunction requires crossovers, which are formed as products of meiotic recombination. For the past two decades, studies of yeast meiosis have provided invaluable insights into evolutionarily conserved mechanisms of meiosis.
BIOLOGY OF YEAST MEIOSIS
Meiosis in budding yeast is an integral part of the differentiation program that results in formation of spores, a dormant cell type that provides resistance against many types of environmental stress. Yeast meiosis is induced by deprivation of nutrients, in particular nitrogen and a fermentable carbon source. This condition relieves glucose-dependent repression of Ime1, which promotes transcription of early meiosis-specific genes (Kassir et al. 1988). Despite its specialized biological function, evolutionary conservation of the meiotic program makes yeast an attractive model system (e.g., Caryl et al. 2000; Kumar et al. 2010). SPECIAL FEATURES OF MEIOSIS Chromosome Segregation
During meiosis, one round of genome duplication is followed by two rounds of chromosome segregation. During the first segregation or meiosis I (MI), homologous chromosomes separate, resulting in halving of the ploidy from diploid to haploid (Fig. 1A). The second meiotic division or meiosis II (MII) is equational, where sister chromatids separate. As a result, four haploid nuclei are generated from a single diploid precursor cell. In yeast, each nucleus becomes engulfed by a separate spore wall during sporogenesis. All four haploid spores, the products of a single meiotic event, remain contained as a tetrad within a common cell wall structure called ascus. 3
Correspondence: [email protected]; [email protected]
© 2015 Cold Spring Harbor Laboratory Press Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top077636
970
Downloaded from http://cshprotocols.cshlp.org/ at University of Liverpool Library on November 30, 2015 - Published by Cold Spring Harbor Laboratory Press
Basic Yeast Meiosis Methodology
B
A Mitotic (equational) 2N
2 x 2N
S
Dad Mom
G0
4 x 1N
0 1 Mitotic cycle G1 S
MI (reductional) MII (equational)
Tetrad
Prophase I MI MII
S 2
G2
3
4
5
6
7
8
Sporo 9 10 Time (h)
M
0 20 40 60 80 100
Time (min)
(ii)
C
Tel1/Mec1 (i)
Resected DSB break ends
SEI
dHJ
CO MI
rad50S
dmc1Δ Tel1/Mec1 (iii)
FIGURE 1. (A) Mitotic (equational) versus meiotic divisions. (S) Mitotic or meiotic replication; (MI) meiosis I; (MII) meiosis II. (B) Timeline of key meiotic events in wild-type SK1 compared to the corresponding events in vegetative cells (Sporo) Sporogenesis. (S) Mitotic or Meiotic replication; Meiotic recombination and homolog synapsis occur during prophase I. Images above the timeline are nuclear morphologies of mono-, di-, and tetra-nucleate cells, and an ascusengulfed tetrad, observable before MI, post MI, post MII, and post sporogenesis, respectively. DNA and tubulin are stained in blue and red, respectively. (C ) Key intermediates leading to crossover (CO) formation in meiotic recombination. (i) DSB: double strand break; SEI: single-end invasions; dHJ: double Holliday junctions (Schwacha and Kleckner 1997; Hunter and Kleckner 2001). (ii) Tel1 and Mec1 are activated by DSBs and regulate Spo11-activity to maintain DSB-homeostasis and ensure IH-bias (Carballo et al. 2008, 2013). (iii) Accumulation of recombination intermediates in rad50S and dmc1Δ background, respectively, trigger Tel1/Mec1-dependent meiotic arrest and/or delay (Lydall et al. 1996; Usui et al. 2001).
Meiotic S Phase
In all organisms examined to date, several stages of meiosis occupy substantially longer time intervals than the corresponding mitotic counterparts (Fig. 1B). For example, meiotic and mitotic S-phases in budding yeast last 60–80 min and 15–20 min, respectively (Williamson et al. 1983; Cha et al. 2000). Although the rate of replication fork progression and the number of replication origins used are comparable in meiotic versus mitotic replication, delayed firing of many replication origins appears to contribute to the prolongation of meiotic S-phase (Collins and Newlon 1994; Blitzblau et al. 2012). Meiotic Recombination
In most organisms, meiotic recombination is initiated after S-phase by Spo11 catalysis of DNA double-strand breaks (DSBs) (Bergerat et al. 1997; Keeney et al. 1997). Recombinational repair of meiotic DSBs proceeds with an interhomolog (IH) bias, whereby the majority of breaks are repaired using an intact nonsister homologous chromosome as a template. IH-bias ensures formation of crossovers (COs) essential for homolog segregation (e.g., Schwacha and Kleckner 1997) (Fig. 1C). Meiotic Chromosome Structure
Meiotic recombination takes place in the context of a tripartite proteinaceous structure, called the synaptonemal complex (SC), which intimately juxtaposes homologous chromosomes (Sym et al. 1993). The extent of SC development defines substages of meiotic prophase, where leptotene, Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top077636
971
Downloaded from http://cshprotocols.cshlp.org/ at University of Liverpool Library on November 30, 2015 - Published by Cold Spring Harbor Laboratory Press
G.V. Börner and R.S. Cha
zygotene, pachytene, and diplotene refer, respectively, to stages of minimal, partial, and full synapsis as well as the stage at which the SC has disassembled.
Regulation of Meiotic Progression
Meiotic progression is regulated by factors known to also control the mitotic cell cycle (e.g., cyclindependent kinase [Cdc28] and polo kinase [Cdc5]) (e.g., Wan et al. 2008; Sourirajan and Lichten 2008). In addition, meiosis-specific proteins such as the transcription factor Ndt80 regulate meiotic progression (Xu et al. 1995). Initiation of meiotic recombination activates the conserved ATM/ATR kinases (i.e., Tel1/Mec1 in budding yeast) (Fig. 1C). During unperturbed meiosis, Tel1/Mec1 function to control the extent of Spo11-catalysis and to promote IH-bias (Carballo et al. 2008, 2013). Tel1/ Mec1 also function as bona fide checkpoint regulators, triggering meiotic arrest in response to meiotic recombination defects (Fig. 1C) (Lydall et al. 1996; Usui et al. 2001).
ANALYSIS OF MEIOSIS IN YEAST
In accompanying protocols, we present selected methods for analyzing meiotic products in budding yeast following sporulation on solid medium (Protocol: Analysis of Yeast Sporulation Efficiency, Spore Viability, and Meiotic Recombination on Solid Medium [Börner and Cha 2015a]), preparing highly synchronous meiotic cultures in liquid medium (Protocol: Induction and Analysis of Synchronous Meiotic Yeast Cultures [Börner and Cha 2015b]), and analyzing meiotic recombination and chromosome dynamics in samples collected from synchronous meiotic cultures (Protocol: Analysis of Meiotic Recombination and Homolog Interaction during Yeast Meiosis [Börner and Cha 2015c]). These protocols provide a practical introduction for the novice to the study of yeast meiosis. Additional yeast meiosis methods (e.g., screening for and isolating meiotic mutants) as well as methods for investigating meiosis in other organisms have been published elsewhere (Guthrie and Fink 1991; Keeney 2009a,b).
Choice of Strain Background
Several strain backgrounds have been used for meiotic analysis (e.g., Cotton et al. 2009; Elrod et al. 2009). However, the methods described in the above-mentioned protocols are optimized for SK1, which is a readily sporulating strain of Saccharomyces cerevisiae. The SK1 strain completes both meiotic divisions by 10 h after transfer to sporulation medium (SPM) at 30˚C (Padmore et al. 1991). Spore formation is observed in >90% of cells within 24 h and the spores typically show >90% viability (e.g., Padmore et al. 1991).
Synchronous Meiotic Cultures
Induction of synchronous meiosis entails a presporulation growth protocol that generates a large number of G0-arrested cells, which are subsequently transferred to liquid SPM (see Protocol: Induction and Analysis of Synchronous Meiotic Yeast Cultures [Börner and Cha 2015b]). In a typical synchronous culture at 30˚C, 50% of active cells have entered meiotic S-phase by 2.5 h, followed by completion of S-phase 1 h later (Padmore et al. 1991; Cha et al. 2000). Meiotic recombination is initiated after S-phase, with 50% of DSBs formed at 4 h and exit from this stage at 5 h. The 50% entry time for appearance of COs and NCOs is 6 h, and the first meiotic division ensues at 7 h (Fig. 1B) (Padmore et al. 1991). Some optimization may be required to obtain a highly synchronous SK1 meiotic culture, with variability attributable to factors such as water quality, temperature, pH, aeration, and pre-SPM growth conditions (e.g., Cha et al. 2000; Börner et al. 2004). 972
Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top077636
Downloaded from http://cshprotocols.cshlp.org/ at University of Liverpool Library on November 30, 2015 - Published by Cold Spring Harbor Laboratory Press
Basic Yeast Meiosis Methodology
Spore Formation and Spore Viability
Most mutations that affect meiosis result in either reduced spore formation and/or reduced spore viability. Reduced sporulation efficiency (see Protocol: Analysis of Yeast Sporulation Efficiency, Spore Viability, and Meiotic Recombination on Solid Medium [Börner and Cha 2015a]) may result from defects in positive drivers of meiotic progression, for example, Ndt80 or Cdc5 (see above). Alternatively, failure to complete meiosis could be due to a Tel1/Mec1-mediated recombination checkpoint response. In the latter case, introduction of mutations that prevent initiation of meiotic recombination (e.g., spo11) or abolish checkpoint response (e.g., MEC1-activating protein Rad24) restores sporulation efficiency (Lydall et al. 1996). Effects of mutations on spore viability can be substantial (
