How can genes be recombined during meiosis

Figure 2: Structure of the Holliday junction. A Electron-microscope image of a recombination intermediate. In this image, the Holliday junction was partially denatured to assist its visualization.

B Two possible configurations for the Holliday junction, with the DNA shown in the parallel left or antiparallel configuration right. Potter, H. DNA recombination: in vivo and in vitro studies. Cold Spring Harb. All rights reserved. Liu, Y.

Happy Hollidays: 40th anniversary of the Holliday junction. Nature Reviews Molecular Cell Biology 5 , Figure Detail.

Although common, genetic recombination is a highly complex process. It involves the alignment of two homologous DNA strands the requirement for homology suggests that this occurs through complementary base-pairing , but this has not been definitively shown , precise breakage of each strand, exchange between the strands, and sealing of the resulting recombined molecules. This process occurs with a high degree of accuracy at high frequency in both eukaryotic and prokaryotic cells. The basic steps of recombination can occur in two pathways, according to whether the initial break is single or double stranded.

In the single-stranded model , following the alignment of homologous chromosomes, a break is introduced into one DNA strand on each chromosome, leaving two free ends. Each end then crosses over and invades the other chromosome, forming a structure called a Holliday junction Figure 2. The next step, called branch migration , takes place as the junction travels down the DNA. The junction is then resolved either horizontally, which produces no recombination, or vertically, which results in an exchange of DNA.

In the alternate pathway initiated by double-stranded breaks, the ends at the breakpoints are converted into single strands by the addition of 3' tails. These ends can then perform strand invasion, producing two Holliday junctions. From that point forward, resolution proceeds as in the single-stranded model Figure 3. Note that a third model of recombination, synthesis-dependent strand annealing [SDSA], has also been proposed to account for the lack of crossover typical of recombination in mitotic cells and observed in some meiotic cells to a lesser degree.

No matter which pathway is used, a number of enzymes are required to complete the steps of recombination. The genes that code for these enzymes were first identified in E. This research revealed that the recA gene encodes a protein necessary for strand invasion. Meanwhile, the recB , recC , and recD genes code for three polypeptides that join together to form a protein complex known as RecBCD; this complex has the capacity to unwind double-stranded DNA and cleave strands.

Two other genes, ruvA and ruvB , encode enzymes that catalyze branch migration , while Holliday structures are resolved by the protein resolvase , which is product of the ruvC gene. In eukaryotes, recombination has been perhaps most thoroughly studied in the budding yeast Saccharomyces cerevisiae.

Many of the enzymes identified in this yeast have also been found in other organisms, including mammalian cells. Such studies reveal that the Rad genes named for the fact that their activity was found to be sensitive to radiation play a key role in eukaryotic recombination.

In particular, the Rad51 gene, which is homologous to recA , encodes a protein called Rad51 that has recombinase activity. This gene is highly conserved, but the accessory proteins that assist Rad51 appear to vary among organisms.

For example, the Rad52 protein is found in both yeast and humans, but it is missing in Drosophila melanogaster and C. RPA has a higher affinity for ssDNA than Rad51, and it therefore can inhibit recombination by blocking Rad51's access to the single strand needed for invasion. Once access has been gained, Rad51 polymerizes on the DNA strand to form what is called a presynaptic filament, which is a right-handed helical filament containing six Rad51 molecules and 18 nucleotides per helical repeat.

The search for DNA homology and formation of the junction between homologous regions is then carried out within the catalytic center of the filament. In addition to proteins that assist Rad51 activity, there are also some proteins that inhibit it. It is thought that these proteins play a role in preventing recombination during DNA replication when it is not needed.

Spo11 oligo density within bp bins starting from the centromere and moving up to 75 kb away, averaged across the 32 chromosome arms was determined. The horizontal dotted line indicates genome average.

Spo11 oligo counts smoothed with a bp Hann window are shown. The black circle indicates the centromere, filled triangles indicate the midpoints of coordinates where RFP red and GFP green cassettes were targeted to for CEN8 analysis in the live cell recombination assay; open triangles indicate the locations where the cassettes were targeted to for ARM8 analysis. The DSB effect varied per individual chromosome Figure 3—figure supplement 1.

The variability in susceptibility of the different pericentromeres to DSBs is likely explained by their underlying features, since it is well-established that DSB formation is influenced by chromatin and genome organization e. Screening additional Ctf19 complex subunits showed a striking correlation between increased DSB formation at CEN1 and increased CO formation as measured in our live cell recombination reporter assay Figure 4B , Figure 4—figure supplement 1A. Interestingly, depletion of the MIND complex component, Dsn1 Figure 1—figure supplement 1 also resulted in the appearance of CEN1 -proximal DSBs, suggesting that the overall integrity of the kinetochore might be generally important for repressing DSB formation within pericentromeres.

In conclusion, one likely mechanism by which the Ctf19 complex prevents pericentromeric recombination is via the inhibition of DSB formation close to centromeres.

Spooligo counts RPM were smoothed with bp Hann window. Arrowheads, Spodependent DSBs. During mitotic growth, the Ctf19 complex targets loading of the sister-chromatid-linking complex, cohesin, to the centromere prior to S phase to enrich cohesin in the surrounding pericentromere Eckert et al. This enrichment provides the basis for robust sister chromatid cohesion, the establishment of which is coupled to DNA replication in S-phase Eckert et al.

Since cohesin has been implicated in influencing meiotic DNA break formation and repair Ellermeier and Smith, ; Klein et al. To prevent pleiotropic phenotypes and sporulation failure associated with total cohesin loss in meiosis, we employed a mutation in the Scc4 subunit of the cohesin loader scc4-m35 , which in vegetative cells specifically abolishes pericentromeric cohesin enrichment Hinshaw et al. In meiotic prophase, Rec8 levels were indeed reduced at centromeric and pericentromeric sites in scc4-m35 cells.

However, chromosomal arm sites were also affected Figure 5E , suggesting that the scc4-m35 mutations might influence cohesin loading at non-centromeric sites during meiosis. Nevertheless, scc4-m35 cells underwent meiosis to produce spores, analysis of which revealed an increased frequency of pericentromeric Figure 5F , but not chromosomal arm Figure 5G COs, though this increase was more modest than in the absence of Ctf19 complex subunits Figure 1B,C.

This finding supports the notion that pericentromeric cohesin enrichment by the Ctf19 complex contributes to the suppression of centromere-proximal COs. A—D , The Ctf19 complex enriches meiotic cohesin in the pericentromere during prophase I. Rec8 association with chromosome V and a close up of the 50 kb pericentromeric interval is shown A.

E Chromosomal Rec8 levels are reduced in scc4-m35 cells. F, G Map distances in cM in the pericentromere F or a chromosome arm G interval in wild type, a control SCC4 replacement strain, or the scc4-m35 mutant were determined and their significance analysed as described in Figure 1. ChIP-Seq, chromatin immunoprecipitation with sequencing. We next asked whether the Ctf19 complex influences DSB patterns near centromeres by acting before and during S-phase, when it is known to promote the pericentromeric enrichment of cohesin.

Next, we examined the effect on centromeric cohesin. Because this assay does not allow cohesin loaded before or after DNA replication to be distinguished, we sought to test the functionality of pericentromeric cohesin in the two conditions.

Since only functional cohesin is expected to be retained at centromeres during anaphase I Klein et al. Therefore, the presence of Ctf19 before and during S phase allows for the establishment of functional pericentromeric cohesin. Having established conditions that allowed us to uncouple pericentromeric cohesin establishment from post-S phase functions of the Ctf19 complex, we asked whether the role of the Ctf19 complex in suppressing pericentromeric DSBs is linked to its role in cohesin establishment during S phase.

DSBs were also observed following rapamycin addition at 3 hr, i. This suggests that the Ctf19 complex is required throughout meiotic prophase to prevent pericentromeric DSB formation and that it does so in a manner independent of its role in cohesin establishment. A, B Scheme of the anchor away system and experimental setup used to deplete Ctf19 during meiosis. C Addition of Rapamycin leads to Ctf19 removal from the pericentromere. See Figure 5 and Supplementary file 4B for details of primer sets used.

E Examples of binucleate cells with centromeric or no Rec8. F Percentages of binucleate cells with centromeric Rec8 are shown for indicated conditions.

G DNA replication is largely complete prior to anchoring Ctf19 away in cultures where Rapamycin was added at 3 hr. Thus, DSB inhibition near centromeres does not depend on cohesin, but requires the continuous presence of the Ctf19 complex. These findings provide an explanation for our observation that Ctf19 complex mutants exhibit a higher frequency of pericentromeric COs than scc4-m35 mutant cells Figure 1C , Figure 5F , despite a comparable reduction in pericentromeric cohesin Figure 5D,E.

We therefore speculate that the large multi-subunit Ctf19 complex may exert DSB suppression near centromeres by altering local chromosome structure such that accessibility of DSB-promoting factors is prevented.

Our findings suggest that pericentromeric cohesin might provide a safeguarding mechanism to channel residual centromere-proximal DNA breaks towards repair pathways that do not promote CO formation. If this is the case, DSBs arising after DNA replication and pericentromeric cohesion establishment would not be expected to give rise to pericentromeric COs.

Therefore, pericentromeric cohesin acts at a step after DSB formation to direct repair, probably through a pathway avoiding the homolog, to ensure CO suppression near centromeres. Because the cohesin complex is known to promote inter-sister recombination in mitosis and meiosis Covo et al. Rec8 globally influences the localization of the synaptonemal complex SC component Zip1 along chromosomes Chuong and Dawson, ; Brar et al. Because Zip1, like cohesin Kim et al.

Nevertheless, ChIP-Seq of prophase I-arrested cells confirmed that Ctf19 complex components are specifically required for Zip1 association with core centromeres and the pericentromere Figure 7D—F. We measured comparable pericentromeric CO frequencies of 2. A Zip1 enrichment on chromosomes is reduced in scc4-m35 mutants.

B-—F The Ctf19 complex is required for Zip1 localization at centromeres. C Analysis of Zip1 localization on chromosome spreads as cells progress into prophase I. Categories of Zip1 localization were scored in spread nuclei at each of the indicated times after resuspension in sporulation medium.

D Zip1 localization along chromosome V is shown as an example with the 50 kb region around the centromere amplified. Centromeric Zip1 mediates homology-independent pairing of homologous chromosomes early in meiotic prophase; the subsequent conversion to homologous pairing requires Spo11 Tsubouchi and Roeder, ; Tsubouchi et al. To test whether Zip1 exerts its role in suppression of pericentromeric COs through homology-independent centromere coupling, we analyzed the synaptonemal complex assembly-proficient but centromere coupling-defective zip1-S75E mutant Falk et al.

Pericentromeric COs were not significantly increased in the zip1-S75E mutant Figure 8E,F , indicating that Zip1 suppresses pericentromeric COs independently of its role in centromere coupling.

The number of Ndc10 kinetochore foci per spread nucleus was scored in the indicated strains at the 6 hr time point with the average and standard deviation indicated. B The inset shows NdcHA staining red in an example nucleus. DNA is shown in blue. A representative experiment is shown. E, F The centromere-coupling function of Zip1 is separable from its role in suppression of recombination in the pericentromere.

COs, crossovers. Centromere-proximal CO recombination is a major risk factor for meiotic chromosome segregation and developmental aneuploidy and is suppressed in species with diverse centromere organization. The data presented here establish the highly conserved kinetochore as a major factor responsible for setting up a repressive environment for crossover recombination in the pericentromere during meiosis Figure 9.

In cells lacking the Ctf19 kinetochore sub-complex, pericentromeric CO formation drastically increases. We uncover multi-layered Ctf19 complex-dependent suppression of CO formation acting at both the level of meiotic DSB formation and at the level of recombinational repair template choice. This centrally important role of the kinetochore in preventing potentially detrimental crossovers nearby defines a further mechanism by which it safeguards genome stability.

The placement of meiotic DSBs is influenced by factors acting on different levels of chromosome and chromatin organization. On a global scale, the assembly of a meiotic chromosome axis dictates the spatial distribution of the meiotic DSB machinery along chromosomes Kim et al. On a smaller scale, genome organization and histone modifications have been shown to allow Spo11 activity reviewed in de Massy Spodependent DSB formation is strongly associated with chromatin regions that are enriched for histone H3 Lysine 4 H3K4 methylation reviewed in de Massy and in budding yeast, these modifications are found near transcriptional start sites Tischfield and Keeney, Indeed, within the budding yeast genome, Spo11 prefers to cleave DNA in open, nucleosome-free regions that are most often found in active, divergent promoters Blitzblau et al.

Superimposed on these global determinants of the DSB landscape are specific and spatial controls, which create zones of inhibition within at-risk genomic regions such as telomeres, repetitive DNA arrays and centromeres reviewed in de Massy The underlying genome organization of these regions is not obviously different from the rest of the genome, harboring a similar density of genes and regulatory elements, which would ordinarily be expected to contain several DSB-permissive regions, were they not located close to a centromere.

The Ctf19 complex suppresses DSB formation surrounding all centromeres, though to different extents depending on the chromosome Figure 3—figure supplement 1 , suggesting that it overcomes the intrinsic features of the chromatin organization in the pericentromere to dampen DSB activity.

Therefore, the Ctf19 complex protects a region about 50 times larger than that occupied by the centromeric nucleosome from DSBs, suggesting that the effect is not merely due to a local disturbance of Cse4 CENP-A. A precedent for the idea that the Ctf19 complex can exert long-range effects on the pericentromere through its role in driving cohesin loading at the centromere to enrich the surrounding 20—50 kb is well-documented Eckert et al.

However, we found that the Ctf19 complex must prevent DSB formation independently of its role in promoting cohesin enrichment within pericentromeres. Taken together, these findings suggest the existence of an additional, distinct Ctf19 complex-dependent effect on meiotic DSB formation within regions adjacent to centromeres.

By analogy to the effect on pericentromeric cohesin enrichment, we speculate that the Ctf19 complex may enable the centromeric recruitment of factors that alter chromatin organization in the surrounding region. Minimizing the initiating event of meiotic recombination, DSB formation, is an efficient way to shield against unwanted pericentromeric CO formation.

However, the prevention of pericentromeric DSBs by the Ctf19 complex is not absolute, as DSBs are observed near centromeres in wild type cells, although at reduced levels Blitzblau et al. We found that DSBs that escape the repressive control of the Ctf19 complex are diverted from repair pathways that would lead to potentially deleterious CO formation by cohesin, which is established at high levels within pericentromeres.

These observations are in agreement with previous conclusions that pericentromeric CO formation is more strongly suppressed than DSB formation Blitzblau et al.

We found that forced removal of the Ctf19 complex from kinetochores after S-phase triggered increased DSB formation, but led to only a relatively modest increase in CO formation. Why Science Matters. The Beyond. Plant ChemCast. Postcards from the Universe. Brain Metrics. Mind Read. Eyes on Environment. Accumulating Glitches. Saltwater Science. Microbe Matters. You have authorized LearnCasting of your reading list in Scitable. Do you want to LearnCast this session?

This article has been posted to your Facebook page via Scitable LearnCast. Change LearnCast Settings. Scitable Chat. Register Sign In. Maybe you have the same nose as your brother or red hair like your mother? Family similarities occur because we inherit traits from our parents in the form of the genes that contribute to the traits. This passing of genes from one generation to the next is called heredity. Simple organisms pass on genes by duplicating their genetic information and then splitting to form an identical organism.

More complex organisms, including humans, produce specialised sex cells gametes that carry half of the genetic information, then combine these to form new organisms. The process that produces gametes is called meiosis. During meiosis in humans, 1 diploid cell with 46 chromosomes or 23 pairs undergoes 2 cycles of cell division but only 1 round of DNA replication.

The result is 4 haploid daughter cells known as gametes or egg and sperm cells each with 23 chromosomes — 1 from each pair in the diploid cell. At conception, an egg cell and a sperm cell combine to form a zygote 46 chromosomes or 23 pairs. This is the 1st cell of a new individual. The halving of the number of chromosomes in gametes ensures that zygotes have the same number of chromosomes from one generation to the next.

This is critical for stable sexual reproduction through successive generations.