We study the mechanism and regulation of meiotic recombination in mice. Parts of this work are part of a long-term collaboration withMariji Jasin Laboratoryin the Developmental Biology Program at Memorial Sloan Kettering Cancer Center.
Landscape exploration breaks double mouse threads
DSBs are more likely to occur in some regions of the genome than others. Understanding the mechanisms that shape the "landscape" of DSBs requires identifying the biochemical factors involved, understanding how they affect Spo11, and deciphering their interactions. We developed a powerful method to solve these problems: each Spo11 oligo is a marker that records the exact location of the break, so oligo deep sequencing allowed us to quantitatively map DSBs in the yeast genome with nucleotide resolution with high sensitivity. We recently extended this methodology to mice (Lange et al. 2016; Yamada et al. 2017). An important hotspot determinant in most mammals is PRDM9, which has a rapidly evolving histone methyltransferase module and a Zn-finger DNA-binding domain. DNA binding specificity defines foci, but it is not known how PRDM9 targets SPO11 activity. Furthermore, although the mammalian DSB landscape is patterned hierarchically at different size scales, as with yeast, research has focused almost exclusively on milestones, so we know little about other characteristics. The much higher resolution and sensitivity of SPO11-oligo mapping compared to previous ssDNA sequencing (SSDS) maps allows us to tackle many of these challenges in ways that were previously not possible.
The role of ATM in the feedback control of double-strand break formation
ATM is a mutated Ser/Thr kinase in the cancer-prone disease, ataxia telangiectasia (AT). DSB-activated ATM activates cell cycle checkpoints and promotes DNA repair in somatic cells, but ATM is also essential during normal, uninterrupted meiosis for unclear reasons.
The breakthrough came with our discovery thatATM–/–spermatocytes have a very large number of DSBs. Furthermore, the lack of ATM makes DSB formation extremely sensitive to SPO11 expression level, which probably explains whySpo11heterozygosity partially savesATMCons: Lots of consATMNull spermatocytes are likely due to significantly elevated DSBs, which are downregulated by reduced SPO11 expression. We proposed that ATM activation works in a negative feedback loop that constrains SPO11 to limit the number of DSBs (Lange et al., 2011). Independent studies inDrosophila(McKim lab) and yeast (Kleckner, Neale, Cha and Fung labs) came to a similar conclusion. 2017).
Sex chromosome segregation is a challenge for the male meiotic cell because X and Y share only a small region of homology, the pseudoautosomal (PAR) region: at least one DSB must arise within a PAR; that DSB needs to locate and involve its partner; and there should be an intersection. To understand how cells deal with these challenges, we studied the behavior and structure of PARs in normal meiosis and investigated the genetic requirements that ensure PAR DSB formation (Kauppi et al. 2011). We found that mouse DNA PAR occupies unusually long chromosomal axes, potentially as shorter chromatin loops to promote DSB formation. We also found that most PARs have late-onset RAD51 outbreaks that mark the end of DSBs and provide evidence that PAR DSBs are genetically distinct from "global" DSBs. These findings reveal specific mechanisms that overcome the unique challenges of X-Y recombination.
Many questions remain, including: What aspects of PAR structure and behavior are inherent in PAR itself? Are pro-DSB agents especially enriched in PAR in men? One approach we use to answer these questions is to query by immuno-FISH about the higher-order structure and composition of PAR proteins in male and female meiosis. In females, the X chromosomes synapse and recombine along their length as autosomes, and PAR has a much lower crossover rate than males. Quantification of RAD51 PAR foci in oocytes will allow us to determine whether less frequent mating reflects less DSB, less chance of DSB becoming mating, or both. Measuring the size of the spindle and loop will test the correlation between chromosome structure and DSB potential. Immuno-FISH will also allow us to test the hypothesis that PAR preferentially accumulates DSB-promoting factors. These and other studies of PAR versus autosomes and spermatocytes versus oocytes will provide critical information on this important region of the genome and help us understand the mechanisms used by meiotic cells to promote precise sex chromosome segregation.
Non-allelic homologous recombination
Recombination usually occurs between allelic sequences in homologs or between sister chromatids, but non-allelic homologous recombination (NAHR) between repeated sequences can result in chromosomal rearrangements. in peopleThe NAHR germline is responsible for more than 30 types of disease-causing mutations, with phenotypes ranging from mental retardation to male infertility. Little is known about this process: What is the mechanism of meiotic NAHR and how does it differ from allelic recombination? What strategies do cells use to limit NAHR? From SPO11-oligo mouse maps, we found several replicates containing strong DSB hotspots (Yamada et al. 2017; S. Kim and J. Lange, unpublished data). We use PCR-based sperm DNA physical assays to investigate the mechanism and regulation of the NAHR by comparing allelic and non-allelic recombination frequencies and testing the hypothesis that the mismatch repair mechanism inhibits the NAHR. This research will provide new insights into how cells reduce the risk of germline genome instability resulting from the need to distribute recombination events in a genome full of repetitive elements.
Discovery of new meiotic genes in mice
Aneuploidy (abnormal number of chromosomes) in gametes (sperm or egg cells) is a major genetic cause of miscarriage and birth defects and is often attributed to errors in chromosome segregation during meiosis. Reverse genetic studies of meiosis in mice have elucidated how meiotic disorders can cause chromosome segregation errors. However, our molecular understanding of key meiotic processes and their contribution to the production of euploid gametes is far from complete. A critical challenge is the lack of knowledge about the complete set of mammalian meiotic genes. To fill this gap, we continue our multi-year efforts to identify candidate genes that may be important for meiosis, generate loss-of-function mutations, and characterize the reproductive phenotypes of mutant mice. For example, we defined an unexpected role in spermiogenesis for BAZ1A (also known as ACF1), the defining subunits of the chromatin remodeling complex of the ISWI ACF family (Dowdle et al. 2013). Current research aims to understand the function of ACF in round spermatids. In current research, we continue to identify and mutate additional candidate genes.
However, it should be noted that orthology-based reverse genetics alone will never be able to fully catalog mammal-specific genes or genes whose sequences have diverged to the point of dilution of homology with known meiotic genes in model organisms from other taxa. Therefore, we also use phenotype-directed chemical mutagenesis in mice to discover new genes important for germline genome stability. Because screening involves random point mutagenesis, it can also generate non-null alleles that reveal underestimated gene functions. Our screening revealed genes involved in meiotic recombination, higher order meiotic chromosomal structure formation (e.g., synaptonemal complex (SC)) and/or meiotic sex chromatin inactivation (MSCI). We recently reported on two new successes of this display:Dnmt3c, which encodes a previously uncharacterized DNA methyltransferase unique to rodents that controls the expression of a transposable element in the male germline (Jain et al. 2017); this isYthdc2, which encodes an RNA helicase that plays a key role in regulating the transition from mitosis to meiosis in the germline (Jain et al. 2018). In the current work, we continue to study the mechanism of action of DNMT3C and YTHDC2 and investigate additional hits that appeared on the screen.
When recombination occurs during meiosis, the cell's homologous chromosomes line up extremely close to one another. Then, the DNA strand within each chromosome breaks in the exact same location, leaving two free ends. Each end then crosses over into the other chromosome and forms a connection called a chiasma.Why is meiotic recombination so important? ›
The study of homologous recombination has its historical roots in meiosis. In this context, recombination occurs as a programmed event that culminates in the formation of crossovers, which are essential for accurate chromosome segregation and create new combinations of parental alleles.Is recombination associated with meiotic division in animals? ›
Most significant percentage of recombination occurs naturally. In eukaryotes, genetic recombination happens during meiosis, where homologous chromosomes are paired. This could be followed by chromosome-to-chromosome information transfer.What are the advantages of recombination during meiosis there are two correct answers? ›
Not only is recombination needed for homologous pairing during meiosis, but recombination has at least two additional benefits for sexual species. It makes new combinations of alleles along chromosomes, and it restricts the effects of mutations largely to the region around a gene, not the whole chromosome.Is meiotic recombination the same as crossing over? ›
Recombination of genes in the gametes is a result of crossing over, leading to variations among the offspring. Both of these events occur in prophase 1 of meiosis 1 in eukaryotes.What is initiation of meiotic recombination? ›
Meiotic recombination is initiated by the formation of DNA double-strand breaks (DSBs) catalyzed by the evolutionary conserved Spo11 protein and accessory factors.What happens to a cell in the absence of meiotic recombination? ›
In this case meiotic recombination is vital: in its absence homologs missegregate and the resulting aneuploid gametes give rise to defective or inviable progeny.What is the purpose of recombination? ›
Recombination is a process by which pieces of DNA are broken and recombined to produce new combinations of alleles. This recombination process creates genetic diversity at the level of genes that reflects differences in the DNA sequences of different organisms.Why is recombination important for species? ›
Genetic recombinations provide a constant DNA homogenization within the species and, therefore, the species integrity as an elementary structure responsible for the preservation and rise in the level of ecological stability of organisms in evolving lineages.What stage is meiotic recombination? ›
Recombination Occurs During the Prolonged Prophase of Meiosis I. Prophase I is the longest and arguably most important segment of meiosis, because recombination occurs during this interval. For many years, cytologists have divided prophase I into multiple segments, based upon the appearance of the meiotic chromosomes.
Most recombination occurs naturally and can be classified into two types: (1) interchromosomal recombination, occurring through independent assortment of alleles whose loci are on different but homologous chromosomes (random orientation of pairs of homologous chromosomes in meiosis I); & (2) intrachromosomal ...What is mitotic and meiotic recombination? ›
In mitotic cells, recombination occurs most frequently between sister chromatids, which act as a template for DNA repair. By contrast, crossing over in meiotic cells is targeted between homologs. This bias is key because only interhomolog crossovers promote proper segregation at meiosis I.What are 3 ways that meiosis allows for genetic recombination? ›
Crossing over (in prophase I) Random assortment of chromosomes (in metaphase I) Random fusion of gametes from different parents.How many times does recombination occur during meiosis? ›
Thus recombinations occur in every meiosis, resulting in at least one exchange between pairs of homologous chromosomes per meiosis.Does meiotic recombination require a double strand break? ›
During meiosis, cells deliberately form numerous DNA double-strand breaks (DSBs) in order to initiate homologous recombination, which exchanges genetic information between homologous chromosomes and promotes accurate chromosome segregation.Does meiotic recombination occur between sister chromatids? ›
Meiotic recombination has two key functions: the faithful assortment of chromosomes into gametes and the creation of genetic diversity. Both processes require that meiotic recombination occurs between homologous chromosomes, rather than sister chromatids.What is meiotic recombination checkpoint in meiosis? ›
In response to incomplete recombination, the pachytene checkpoint (also known as the meiotic recombination checkpoint) arrests or delays meiotic cell cycle progression, thus preventing the formation of defective gametes.What are two main causes of recombination? ›
- Recombination occurs when the chromosomes break at the same locus in the base pair sequence which results in the exchange of genes.
- Recombination during meiosis is caused by crossing over. ...
- The chiasma breaks and the broken chromosome segments get switched onto homologous chromosomes leading to recombination.
In this instance, the outcome of recombination is to ensure that each gamete includes both maternally and paternally derived genetic information, such that the resulting offspring will inherit genes from all four of its grandparents, thereby acquiring a maximum amount of genetic diversity.What is recombination and how is initiated and when and why? ›
Meiosis involves a DNA recombination step between parental chromosomes. Recombination is initiated by a DNA double-strand break, which can exchange DNA between the chromosomes, a process that drives human genetic variation.
The effects of recombination can be good, as it can facilitate adaptation, but also bad when it breaks apart beneficial combinations of alleles, and recombination is highly variable between taxa, species, individuals and across the genome.How does recombination impact a species variation? ›
By breaking up linkage, recombination makes it easier for natural selection to target individual genes while avoiding the potentially disadvantageous effect of simultaneously reducing diversity at neighboring genes (a phenomenon known as “Hill-Robertson interference”).Does recombination increase genetic diversity? ›
In regions of high recombination, the footprint is expected to be smaller because recombination moves the beneficial mutation onto different genetic backgrounds, allowing linked diversity to persist.What is meiotic recombination in humans? ›
A central mechanism of meiosis is recombination between homologous chromosomes, during which programmed DNA double-strand breaks (DSBs) are sequentially repaired to form the crossovers essential for faithful chromosomal segregation.Where does meiotic recombination occur? ›
Meiotic recombination  refers to the reciprocal physical exchange of chromosomal DNA between the parental chromosomes and occurs at meiosis during spermatogenesis and oogenesis, serving to ensure proper chromosome segregation.
Mitotic recombination is often initiated by single-stranded DNA (ssDNA), which can arise by several avenues (Mehta and Haber 2014). They include the processing of DNA double-strand breaks by 5′ to 3′ resection, during replication of damaged DNA, or during excision repair (Symington 2014).What is an example of mitotic recombination? ›
For example, Bloom's syndrome is caused by a mutation in RecQ helicase, which plays a role in DNA replication and repair. This mutation leads to high rates of mitotic recombination in mice, and this recombination rate is in turn responsible for causing tumor susceptibility in those mice.In what step of meiosis would recombinants form and why? ›
Recombinant DNA is made at the pachytene stage during the end of prophase-1. The homologous chromosomes come together physically to cross over and exchange parts of their chromosome. This results in recombination of DNA in homologous chromosomes.What are the three methods of genetic recombination and why it is important? ›
Transduction, transformation, and conjugation are three recombination strategies used by bacteria (prokaryotes) to expand their genetic variety.What are the three types of recombination? ›
There are three types of recombination; Radiative, Defect, and Auger. Auger and Defect recombination dominate in silicon-based solar cells. Among other factors, recombination is associated with the lifetime of the material, and thus of the solar cell.
Such genetic analysis has established that recombination requires specific enzymes, in addition to proteins (such as DNA polymerase, ligase, and single-stranded DNA-binding proteins) that function in multiple aspects of DNA metabolism.Why does recombination only occur when crossing over happens in meiosis I? ›
Crossing over (recombination) only occurs during Prophase 1 of Meiosis because at this point homologous chromosomes line up at the centre of the cell. Thus, the aligned chromosomes are able to have their legs intertwine with that of the chromosome beside them, in order for crossing over to occur.What will happen if there are no crossing over and recombination in the mechanism of meiosis? ›
If a mutation prevents crossing over, exchange of DNA between homologous chromosomes will not occur. This will result in reduced genetic variation in an organism's gametes and ultimately its offspring.What is the process of mitotic recombination? ›
Mitotic recombination is often initiated by single-stranded DNA (ssDNA), which can arise by several avenues (Mehta and Haber 2014). They include the processing of DNA double-strand breaks by 5′ to 3′ resection, during replication of damaged DNA, or during excision repair (Symington 2014).What is the meiotic process that results in genetic recombination? ›
In eukaryotes, recombination during meiosis is facilitated by chromosomal crossover. The crossover process leads to offspring having different combinations of genes from those of their parents, and can occasionally produce new chimeric alleles.What is involved in the process of recombination? ›
Recombination is a process by which pieces of DNA are broken and recombined to produce new combinations of alleles. This recombination process creates genetic diversity at the level of genes that reflects differences in the DNA sequences of different organisms.Where does mitotic recombination occur? ›
At what point in the cell cycle does mitotic recombination occur? Whereas meiotic recombination occurs during meiosis, most mitotic recombination probably does not occur during mitosis, but during interphase.What is mitotic recombination in biology? ›
A process in which a diploid cell undergoing mitosis gives rise to daughter cells with allele combinations different from that in the parental cell.In which stage of meiosis does recombination? ›
Recombination Occurs During the Prolonged Prophase of Meiosis I. Prophase I is the longest and arguably most important segment of meiosis, because recombination occurs during this interval.What are the two processes result in recombination? ›
The crossing over process is when genetic material is exchanged between paired chromosomes to create new genes on each chromosome. Independent assortment is when cells divide so gametes can pair with other chromosomes. Fertilization is when two gametes combine to create an offspring.