Frequent and efficient use of the sister chromatid for DNA double-strand break repair during budding yeast meiosis.
Frequent and Efficient Use of the Sister Chromatid for DNA Double-Strand Break Repair during Budding Yeast Meiosis Tamara Goldfarb, Michael Lichten* Laboratory of Biochemistry and Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America Abstract Recombination between homologous chromosomes of different parental origin (homologs) is necessary for their accurate segregation during meiosis. It has been suggested that meiotic inter-homolog recombination is promoted by a barrier to inter-sister-chromatid recombination, imposed by meiosis-specific components of the chromosome axis. Consistent with this, measures of Holliday junction–containing recombination intermediates (joint molecules [JMs]) show a strong bias towards inter-homolog and against inter-sister JMs. However, recombination between sister chromatids also has an important role in meiosis. The genomes of diploid organisms in natural populations are highly polymorphic for insertions and deletions, and meiotic double-strand breaks (DSBs) that form within such polymorphic regions must be repaired by inter-sister recombination. Efforts to study inter-sister recombination during meiosis, in particular to determine recombination frequencies and mechanisms, have been constrained by the inability to monitor the products of intersister recombination. We present here molecular-level studies of inter-sister recombination during budding yeast meiosis. We examined events initiated by DSBs in regions that lack corresponding sequences on the homolog, and show that these DSBs are efficiently repaired by inter-sister recombination. This occurs with the same timing as inter-homolog recombination, but with reduced (2- to 3-fold) yields of JMs. Loss of the meiotic-chromosome-axis-associated kinase Mek1 accelerates inter-sister DSB repair and markedly increases inter-sister JM frequencies. Furthermore, inter-sister JMs formed in mek1D mutants are preferentially lost, while inter-homolog JMs are maintained. These findings indicate that intersister recombination occurs frequently during budding yeast meiosis, with the possibility that up to one-third of all recombination events occur between sister chromatids. We suggest that a Mek1-dependent reduction in the rate of intersister repair, combined with the destabilization of inter-sister JMs, promotes inter-homolog recombination while retaining the capacity for inter-sister recombination when inter-homolog recombination is not possible. Citation: Goldfarb T, Lichten M (2010) Frequent and Efficient Use of the Sister Chromatid for DNA Double-Strand Break Repair during Budding Yeast Meiosis. PLoS Biol 8(10): e1000520. doi:10.1371/journal.pbio.1000520 Academic Editor: R. Scott Hawley, Stowers Institute for Medical Research, United States of America Received April 15, 2010; Accepted September 2, 2010; Published October 19, 2010 This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Funding: This work was supported by the Intramural Research Program at the National Cancer Institute, National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Abbreviations: BSCR, barrier to sister chromatid recombination; CO, crossover; DSB, double-strand break; IH, inter-homolog; IS, inter-sister; JM, joint molecule; MI, first meiotic division; NCO, noncrossover; SEM, standard error of the mean. * E-mail: email@example.com repair during meiosis is distinct from that during the mitotic cell cycle. During the mitotic cell cycle, there is a strong bias to repair DSBs using the sister chromatid [4,5]. In contrast, the homolog is often used to repair DSBs during meiosis, with two possible outcomes. After initial repair synthesis, the invading strand can detach from the homolog and reanneal with the unresected strand of the second DSB end to form a noncrossover (NCO) product in a process called synthesis-dependent strand annealing . Alternatively, if the second end of the DSB also associates with donor sequences, Holliday junction–containing intermediates, here called joint molecules (JMs), are formed . In budding yeast, these are mostly resolved as COs [8,9]. It is generally thought that IH recombination dominates during meiosis. In budding yeast, most JMs form between parental homologs, with only 13%–25% forming between sister chromatids [7,10–12]. dmc1 mutants fail to repair DSBs and do not form JMs during meiosis, but rapidly form inter-sister (IS) JMs, and not IH JMs, when returned to vegetative growth . In haploid yeast Introduction During meiosis, the diploid genome is reduced to produce haploid gametes through two successive rounds of nuclear division that follow a single round of DNA replication. Homologous parental chromosomes (homologs) pair and separate at the first meiotic division (MI), while sister chromatids segregate during the second division. Crossover (CO) products of inter-homolog (IH) recombination, combined with sister chromatid cohesion, ensure proper chromosome disjunction at MI, and a failure to properly create these connections results in aneuploid progeny. Aneuploidy caused by MI non-disjunction is a leading cause of both miscarriage and congenital birth defects . Meiotic recombination is initiated by double-strand breaks (DSBs) formed by the Spo11 protein . DSBs are resected to form single strands that are substrates for strand invasion catalyzed by the meiosis-specific Dmc1 and the ubiquitous Rad51 proteins . The choice of a target for strand invasion and subsequent PLoS Biology | www.plosbiology.org 1 October 2010 | Volume 8 | Issue 10 | e1000520 Sister Chromatid Recombination in Yeast Meiosis These findings, while consistent with a Mek1-dependent BSCR during meiosis, were obtained under circumstances where repair and recombination are altered genome-wide. In particular, abnormally high levels of unrepaired DSBs in dmc1 mutants and in haploid cells undergoing meiosis may result in altered repair mechanisms and outcomes. For example, the resection and repair of meiotic DSBs formed by the site-specific VDE endonuclease are altered in dmc1 mutants by the presence or absence of other hyperresected Spo11-catalyzed DSBs [27,28]. While it is clear that IS recombination is less prevalent during meiosis than during vegetative growth, knowledge of the relative efficiency of IH and IS recombination during meiosis remains incomplete. Previous studies have inferred the relative frequency of IS and IH repair by comparing IS- and IH-containing JM intermediates. However, no study has directly measured the efficiency of all types of IS repair in normal diploids, partly because such measurements are hampered by the inability to detect many of the products of IS recombination. To address this issue, we monitored the fate of a DSB that could only be repaired by sister chromatid recombination, in cells where all other DSBs could be repaired by IH recombination. We show here that during normal diploid meiosis, such DSBs are efficiently repaired from the sister chromatid. This IS repair has many of the features of normal IH recombination, except that fewer JM intermediates are produced. Based on these and other observations, we suggest that repair from the sister occurs frequently during budding yeast meiosis, even when the homolog is present. We propose that the apparent BSCR is actually a kinetic impediment, imposed by the Mek1 kinase, that roughly equalizes rates of IS and IH recombination during meiosis, a process that would otherwise greatly favor IS events given the spatial proximity of the sister chromatid. Author Summary In diploid organisms, which contain two parental sets of chromosomes, double-stranded breaks in DNA can be repaired by recombination, either with a copy of the chromosome produced by replication (the sister chromatid), or with either chromatid of the other parental chromosome (the homolog). During meiosis, recombination with the homolog ensures faithful segregation of chromosomes to gametes (sperm or egg). It has been suggested that use of the spatially distant homolog, as opposed to the nearby sister chromatid, results from a meiosis-specific barrier to recombination between sister chromatids. However, there are situations where meiotic recombination must occur between sister chromatids, such as when recombination initiates in sequences that are absent from the homolog. By studying such a situation, we show that meiotic recombination with the sister chromatid occurs with similar timing and efficiency as recombination with the homolog. Further analysis indicates that intersister recombination is more common than was previously thought, although still far less prevalent than in somatic cells, where inter-sister recombination predominates. We suggest that meiosis-specific factors act to roughly equalize repair from the sister and homolog, which both allows the establishment of physical connections between homologs and ensures timely repair of breaks incurred in regions lacking corresponding sequences on the homolog. undergoing meiosis, a large fraction of DSBs persist unrepaired, suggesting that IS DSB repair is inefficient [13,14]. These findings have been taken as evidence for a meiosis-specific barrier to sister chromatid recombination (BSCR) that prevents IS recombination and thus promotes IH recombination. The axial element is a structure that forms between sister chromatids early in meiotic prophase. It later becomes part of the synaptonemal complex, a tripartite structure with axes of each homolog closely juxtaposed by transverse filaments . In budding yeast, axial element components Red1 and Hop1, along with the axis-associated, meiosis-specific Mre4/Mek1 kinase (hereafter Mek1), have been suggested as mediating a BSCR [16,17]. Recent studies indicate that meiotic DSBs activate the Mec1 and Tel1 checkpoint kinases, which phosphorylate Hop1 [17,18]. Phosphorylated Hop1 binds and activates the Mek1 kinase, which phosphorylates targets that include the Rad51 accessory factors Rad54 and Rdh54 [19,20]. This prevents interactions between these factors and Rad51 and thus is thought to decrease IS recombination. Evidence consistent with this mechanism is provided by several findings. While DSBs accumulate to normal levels in DSB processing/repair-defective mek1 rad50S double mutants [21,22], mek1 single mutants display reduced steady-state DSB levels and reduced IH COs [21,23], as would be expected if DSBs were rapidly repaired by IS recombination in the absence of axismediated signaling. Consistent with this, both red1 and mek1 mutants display a marked excess of IS JMs over IH JMs [10,24]. Further support for the suggestion that loss of axis signaling allows rapid IS recombination comes from findings that the DSB repair defect of dmc1 mutants is suppressed by hop1, red1, or mek1 loss of function mutations [10,17,19–21,25], and that mek1 suppresses the DSB repair defect seen in haploid yeast undergoing meiosis . Additionally, the meiotic repair defect of dmc1 mutants is partially suppressed by overexpression of RAD51  or RAD54 , and more extensively by overexpression of a RAD54 allele that lacks a Mek1 phosphorylation site . PLoS Biology | www.plosbiology.org Results Meiotic DSBs Are Efficiently Repaired in the Absence of Corresponding Sequences on the Homolog We examined DSBs at two hotspots on chromosome III: within a 3.5-kb recombination reporter construct containing URA3 and ARG4 sequences inserted at HIS4 (his4::URA3-arg4; ) and in the YCR047c promoter (Figure 1A). Both DSB hotspots were examined in a hemizygous configuration, where the hotspot was present on one copy of chromosome III and a small deletion was present on the other homolog. This eliminates the possibility of repair of the hotspot DSB by IH recombination (Figure 1B), but preserves normal homolog alignment, synapsis, and IH recombination in the genome as a whole. We also examined a strain hemizygous for a deletion that removes most of the chromosome III left arm, including sequences for about 45 kb to either side of the his4::URA3-arg4 insertion site (Figure 1B). In most experiments, DSB dynamics were examined in the same strain at a hemizygous site and at a homozygous control site, to control for culture-toculture variation in meiotic progression and the fraction of cells undergoing meiosis. It has been reported that heterozygosity for a small deletion covering a DSB site causes a modest reduction in DSB levels [30,31]. This is not the case for the loci and deletions used here. Cumulative DSB levels at both the his4::URA3-arg4 insert and YCR047c were measured in rad50S mutants, which form, but do not repair, DSBs . DSBs accumulated to similar levels in deletion hemizygotes and in homozygous controls (Figure 1D, right-hand axes). In RAD50 strains, similar DSB dynamics were seen when corresponding sequences were present on or absent from the homolog (Figures 1D and S1). Calculated DSB life spans 2 October 2010 | Volume 8 | Issue 10 | e1000520 Sister Chromatid Recombination in Yeast Meiosis Figure 1. Similar timing of IS and IH DSB repair. (A) Structure of DSB hotspots in a 3.5-kb his4::URA3-arg4 insert  and in the YCR047c– YCR048w intergenic region . White boxes indicate URA3-arg4 insert genes; grey boxes indicate other genes on chromosome III; horizontal bars indicate sequences used for probes [8,29]; vertical lines indicate restriction sites used to detect DSBs (short lines) and JM intermediates (long lines). (B) Strains used. (i) Homozygous control—his4::URA3-arg4 and YCR047c–YCR048w are present on both homologs. (ii) Hemizygous at his4::URA3-arg4— the 3.5-kb his4::URA3-arg4 insert is present on only one homolog. (iii) Left arm deletion—the his4::URA3-arg4 insert is present on one homolog, opposite a 90-kb deletion on the other homolog. (iv) Hemizygous at YCR047c—4 kb of DNA between YCR046c and YCR051w is deleted from one homolog. (C) Southern blots showing detection of DSBs in wild-type and rad50S strains hemizygous for his4::URA3-arg4 at either the his4::URA3-arg4 site (left) or the YCR047c homozygous control site (right). P, parental band; DSB, DSB band. (D) DSB frequencies (3–4 independent experiments, error bars indicate SEM), quantified as percent of total lane signal. Symbols connected by lines are noncumulative DSB frequencies from RAD50 strains (lefthand y-axis); unconnected symbols at 7 h are cumulative DSB frequencies from rad50S strains are (right-hand y-axis). (E) DSB life span, calculated using 7-h rad50S cumulative DSB levels, as described previously . Underlined life spans are for DSBs that must be repaired by IS recombination. Strains in (C–E) are (for RAD50 and rad50S, respectively): homozygous, MJL3201 and MJL3198; hemizygous at his4::URA3-arg4, MJL3250 and MJL3338; left arm deletion, MJL3227 and MJL3233; and hemizygous at YCR047c, MJL3399 and MJL3408. doi:10.1371/journal.pbio.1000520.g001 homolog could also result in repair, by IH gene conversion, leading to loss of the hemizygous sequences. Given DSB frequencies at his4::URA3-arg4 (about 20% of insert-bearing chromosomes), DSB repair by IH gene conversion would result in 36%–40% of tetrads showing 1:3 segregation for the insert. However, only 1% of tetrads from a his4::URA3-arg4 hemizygote showed this segregation pattern (Figure 2B). In summary, all available molecular, meiotic progression, and spore survival data indicate that, when no other homologous repair partners are available at a DSB site, the sister chromatid is used as efficiently for meiotic DSB repair as would be the homolog—at least in strains where most other DSB sites are homozygous and can be repaired by IH recombination. (Figure 1E; see Materials and Methods) at both loci were similar in the presence or absence of homology on the homolog. To confirm that the absence of corresponding homology at one DSB site did not have a chromosome-wide effect on repair, DSB levels were also examined at a DSB site (YFL021w) on chromosome VI. Similar noncumulative DSB curves were observed at this site and at the tester sites on chromosome III (Figure S1D; data not shown). A BSCR-induced delay in DSB repair at a hemizygous site might cause a DNA-damage-response-induced delay in the MI division. We did not observe a significant difference between his4::URA3-arg4 or YCR047c hemizygotes and fully homozygous controls for meiotic division timing or for the fraction of cells that transited meiotic divisions (Figure 2A; data not shown). In addition, no loss of spore viability was observed in strains hemizygous for the his4::URA3-arg4 insert (Figure 2B), as might be expected if an unrepaired DSB persisted through meiotic divisions and sporulation. While these findings are consistent with efficient repair of a DSB in hemizygous sequences by IS recombination, DSB end resection past the region of heterology and subsequent strand invasion of the PLoS Biology | www.plosbiology.org Reduced JM Formation in the Absence of Corresponding Sequences on the Homolog Synthesis-dependent strand annealing, which does not involve Holliday junction–containing intermediates, is thought to be the predominant mechanism of DSB repair during the S and G2 phases of the mitotic cell cycle [5,33,34] and for NCO formation 3 October 2010 | Volume 8 | Issue 10 | e1000520 Sister Chromatid Recombination in Yeast Meiosis This, in turn, indicates that meiotic DSB repair events by IS recombination, when the sister is the only template for repair, produces a equilibrium value of MeCpG steps (,+14 deg.) [31,44]. In comparison, methylation has a significantly lower stability cost when happening at major groove positions, such as 211 and 21 base pair from dyad (mutations 9 and 12), where the roll of the nucleosome bound conformation (+10 deg.) is more compatible with the equilibrium geometry of MeCpG steps. The nucleosome destabilizing effect of cytosine methylation increases with the number of methylated cytosines, following the same position dependence as the single methylations. The multiple-methylation case reveals that each major groove meth- PLOS Computational Biology | www.ploscompbiol.org 3 November 2013 | Volume 9 | Issue 11 | e1003354 DNA Methylation and Nucleosome Positioning ylation destabilizes the nucleosome by around 1 kJ/mol (close to the average estimate of 2 kJ/mol obtained for from individual methylation studies), while each minor groove methylation destabilizes it by up to 5 kJ/mol (average free energy as single mutation is around 6 kJ/mol). This energetic position-dependence is the reverse of what was observed in a recent FRET/SAXS study . The differences can be attributed to the use of different ionic conditions and different sequences: a modified Widom-601 sequence of 157 bp, which already contains multiple CpG steps in mixed orientations, and which could assume different positioning due to the introduction of new CpG steps and by effect of the methylation. The analysis of our trajectories reveals a larger root mean square deviation (RMSD) and fluctuation (RMSF; see Figures S2– S3 in Text S1) for the methylated nucleosomes, but failed to detect any systematic change in DNA geometry or in intermolecular DNA-histone energy related to methylation (Fig. S1B, S1C, S4–S6 in Text S1). The hydrophobic effect should favor orientation of the methyl group out from the solvent but this effect alone is not likely to justify the positional dependent stability changes in Figure 2, as the differential solvation of the methyl groups in the bound and unbound states is only in the order of a fraction of a water molecule (Figure S5 in Text S1). We find however, a reasonable correlation between methylation-induced changes in hydrogen bond and stacking interactions of the bases and the change in nucleosome stability (see Figure S6 in Text S1). This finding suggests that methylation-induced nucleosome destabilization is related to the poorer ability of methylated DNA to fit into the required conformation for DNA in a nucleosome. Changes in the elastic deformation energy between methylated and un-methylated DNA correlate with nucleosomal differential binding free energies To further analyze the idea that methylation-induced nucleosome destabilization is connected to a worse fit of methylated DNA into the required nucleosome-bound conformation, we computed the elastic energy of the nucleosomal DNA using a harmonic deformation method [36,37,44]. This method provides a rough estimate of the energy required to deform a DNA fiber to adopt the super helical conformation in the nucleosome (full details in Suppl. Information Text S1). As shown in Figure 2, there is an evident correlation between the increase that methylation produces in the elastic deformation energy (DDE def.) and the free energy variation (DDG bind.) computed from MD/TI calculations. Clearly, methylation increases the stiffness of the CpG step , raising the energy cost required to wrap DNA around the histone octamers. This extra energy cost will be smaller in regions of high positive roll (naked DNA MeCpG steps have a higher roll than CpG steps ) than in regions of high negative roll. Thus, simple elastic considerations explain why methylation is better tolerated when the DNA faces the histones through the major groove (where positive roll is required) that when it faces histones through the minor groove (where negative roll is required). Nucleosome methylation can give rise to nucleosome repositioning We have established that methylation affects the wrapping of DNA in nucleosomes, but how does this translate into chromatin structure? As noted above, accumulation of minor groove methylations strongly destabilizes the nucleosome, and could trigger nucleosome unfolding, or notable changes in positioning or phasing of DNA around the histone core. While accumulation of methylations might be well tolerated if placed in favorable positions, accumulation in unfavorable positions would destabilize the nucleosome, which might trigger changes in chromatin structure. Chromatin could in fact react in two different ways in response to significant levels of methylation in unfavorable positions: i) the DNA could either detach from the histone core, leading to nucleosome eviction or nucleosome repositioning, or ii) the DNA could rotate around the histone core, changing its phase to place MeCpG steps in favorable positions. Both effects are anticipated to alter DNA accessibility and impact gene expression regulation. The sub-microsecond time scale of our MD trajectories of methylated DNAs bound to nucleosomes is not large enough to capture these effects, but clear trends are visible in cases of multiple mutations occurring in unfavorable positions, where unmethylated and methylated DNA sequences are out of phase by around 28 degrees (Figure S7 in Text S1). Due to this repositioning, large or small, DNA could move and the nucleosome structure could assume a more compact and distorted conformation, as detected by Lee and Lee , or a slightly open conformation as found in Jimenez-Useche et al. . Using the harmonic deformation method, we additionally predicted the change in stability induced by cytosine methylation for millions of different nucleosomal DNA sequences. Consistently with our calculations, we used two extreme scenarios to prepare our DNA sequences (see Fig. 3): i) all positions where the minor grooves contact the histone core are occupied by CpG steps, and ii) all positions where the major grooves contact the histone core are occupied by CpG steps. We then computed the elastic energy required to wrap the DNA around the histone proteins in unmethylated and methylated states, and, as expected, observed that methylation disfavors DNA wrapping (Figure 3A). We have rescaled the elastic energy differences with a factor of 0.23 to match the DDG prediction in figure 2B. In agreement with the rest of our results, our analysis confirms that the effect of methylation is position-dependent. In fact, the overall difference between the two extreme methylation scenarios (all-in-minor vs all-in-major) is larger than 60 kJ/mol, the average difference being around 15 kJ/ mol. We have also computed the elastic energy differences for a million sequences with CpG/MeCpG steps positioned at all possible intermediate locations with respect to the position (figure 3B). The large differences between the extreme cases can induce rotations of DNA around the histone core, shifting its phase to allow the placement of the methylated CpG steps facing the histones through the major groove. It is illustrative to compare the magnitude of CpG methylation penalty with sequence dependent differences. Since there are roughly 1.5e88 possible 147 base pairs long sequence combinations (i.e., (4n+4(n/2))/2, n = 147), it is unfeasible to calculate all the possible sequence effects. However, using our elastic model we can provide a range of values based on a reasonably large number of samples. If we consider all possible nucleosomal sequences in the yeast genome (around 12 Mbp), the energy difference between the best and the worst sequence that could form a nucleosome is 0.7 kj/mol per base (a minimum of 1 kJ/mol and maximum of around 1.7 kJ/mol per base, the first best and the last worst sequences are displayed in Table S3 in Text S1). We repeated the same calculation for one million random sequences and we obtained equivalent results. Placing one CpG step every helical turn gives an average energetic difference between minor groove and major groove methylation of 15 kJ/ mol, which translates into ,0.5 kJ/mol per methyl group, 2 kJ/ mol per base for the largest effects. Considering that not all nucleosome base pair steps are likely to be CpG steps, we can conclude that the balance between the destabilization due to CpG methylation and sequence repositioning will depend on the PLOS Computational Biology | www.ploscompbiol.org 4 November 2013 | Volume 9 | Issue 11 | e1003354 DNA Methylation and Nucleosome Positioning Figure 3. Methylated and non-methylated DNA elastic deformation energies. (A) Distribution of deformation energies for 147 bplong random DNA sequences with CpG steps positioned every 10 base steps (one helical turn) in minor (red and dark red) and major (light and dark blue) grooves respectively. The energy values were rescaled by the slope of a best-fit straight line of figure 2, which is 0.23, to source of circulating FGF-21. The lack of association between circulating and muscle-expressed FGF-21 also suggests that muscle FGF-21 primarily works in a local manner regulating glucose metabolism in the muscle and/or signals to the adipose tissue in close contact to the muscle. Our study has some limitations. The number of subjects is small and some correlations could have been significant with greater statistical power. Another aspect is that protein levels of FGF-21 were not determined in the muscles extracts, consequently we cannot be sure the increase in FGF-21 mRNA is followed by increased protein expression. In conclusion, we show that FGF-21 mRNA is increased in skeletal muscle in HIV patients and that FGF-21 mRNA in muscle correlates to whole-body (primarily reflecting muscle) insulin resistance. These findings add to the evidence that FGF-21 is a myokine and that muscle FGF-21 might primarily work in an autocrine manner. Acknowledgments We thank the subjects for their participation in this study. 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