Biology subjects

Sekelsky, J.

Publications and source records attributed to Sekelsky, J..

15 recordsLinked to original sources

bioRxiv2026

Altering dosage of meiotic crossover-associated RING finger proteins affects crossover number and interference in Drosophila

Crossovers play a critical role in ensuring correct reductional segregation of homologous chromosomes in the first meiotic division. Crossing over is initiated by formation of DNA double-strand breaks (DSBs), but the number of DSBs is greater than the number of crossovers. Which recombination sites become crossovers, versus being repaired as non-crossovers, is not random, but is subject to several crossover patterning phenomena, including crossover assurance and crossover interference. One current model for crossover designation proposes that crossover-associated RING finger proteins (CORs) undergo the biophysical process of coarsening, in which larger accumulations continue to get larger and smaller accumulations go away. Genetic and cytological studies of the three CORs in Drosophila melanogaster, Vilya, Narya, and Nenya, are consistent with this model. In females heterozygous for a deletion of vilya, fewer doublecrossovers are observed. Conversely, crossovers are elevated in females carrying a duplication of vilya and in females coordinately overexpressing Vilya, Narya, and Nenya. These findings support a model in which crossover designation occurs through coarsening of COR proteins within the synaptonemal complex.

genetics↗

bioRxiv2026

Chromosome-specific differences in the recombination landscape of spontaneous meiotic nondisjunction

Failures in chromosome segregation during meiosis result in aneuploid gametes and are the leading cause of miscarriage. The position and number of crossovers, genetic exchanges between homologous chromosomes, are essential to their accurate disjunction in meiosis. Previous research analyzing nondisjunction of acro- and telocentric chromosomes in human and Drosophila has identified altered positioning and number of crossovers that differs between meiosis I and meiosis II nondisjunction. Limited data from positioning in chromosomes that segregated normally in metacentric trisomies has suggested discrepancies between the behavior of these chromosomes and the acro/telocentrics in nondisjunction, which warrants further investigation. Here, we employ whole-genome sequencing to study spontaneous meiotic nondisjunction of the metacentric chromosome 2 in Drosophila melanogaster. Several patterns of recombination may differentially impact telo- versus metacentric chromosomes: lack of a crossover, distal crossovers, and proximal crossovers. We find that unlike meiotic nondisjunction of the Drosophila X chromosome, nondisjunction of chromosome 2 is not associated with dramatic changes in crossover landscape, but is associated with reduced recombination. Differences in the proportions of NDJ events with altered recombination patterns between chromosomes X and 2 suggest that abnormal crossover positions disparately affect chromosomes with different shapes. Taken together, these findings highlight that the underlying triggers of meiotic nondisjunction are chromosome-specific.

genetics↗

bioRxiv2024

Suppression of meiotic crossovers in pericentromeric heterochromatin requires synaptonemal complex and meiotic recombination factors in Drosophila melanogaster

The centromere effect (CE) is a meiotic phenomenon that ensures meiotic crossover suppression in pericentromeric regions. Despite being a critical safeguard against nondisjunction, the mechanisms behind the CE remain unknown. Previous studies have shown that various regions of the Drosophila pericentromere, encompassing proximal euchromatin, beta and alpha heterochromatin, undergo varying levels of crossover suppression, raising the question of whether distinct mechanisms establish the CE in these different regions. To address this question, we asked whether different pericentromeric regions respond differently to mutations that impair various features that may play a role in the CE. In flies with a mutation that affects the synaptonemal complex (SC), a structure is hypothesized to have important roles in recombination and crossover patterning, we observed a significant redistribution of pericentromeric crossovers from proximal euchromatin towards beta heterochromatin but not alpha heterochromatin, indicating a role for the SC in suppressing crossovers in beta heterochromatin. In flies mutant for mei-218 or rec, which encode components of a critical pro-crossover complex, there was a more extreme redistribution of pericentromeric crossovers towards both beta and alpha heterochromatin, suggesting an important role for these meiotic recombination factors in suppressing heterochromatic crossovers. Lastly, we mapped crossovers in flies mutant for Su(var)3-9. Although we expected a strong alleviation of crossover suppression in heterochromatic regions, no changes in pericentromeric crossover distribution were observed in this mutant, indicating that this vital heterochromatin factor is dispensable to prevent crossovers in heterochromatin. Our results indicate that the meiotic machinery plays a bigger role in suppressing crossovers than the chromatin state.

genetics↗

bioRxiv2024

Functions of the Bloom Syndrome Helicase N-terminal Intrinsically Disordered Region

Bloom Syndrome helicase (Blm) is a RecQ family helicase involved in DNA repair, cell-cycle progression, and development. Pathogenic variants in human BLM cause the autosomal recessive disorder Bloom Syndrome, characterized by predisposition to numerous types of cancer. Prior studies of Drosophila Blm mutants lacking helicase activity or protein have shown sensitivity to DNA damaging agents, defects in repairing DNA double-strand breaks (DSBs), female sterility, and improper segregation of chromosomes in meiosis. Blm orthologs have a well conserved and highly structured RecQ helicase domain, but more than half of the protein, particularly in the N-terminus, is predicted to be unstructured. Because this region is poorly conserved across multicellular organisms, we compared closely related species to identify regions of conservation, potentially indicating important functions. We deleted two of these Drosophila-conserved regions in D. melanogaster using CRISPR/Cas9 gene editing and assessed the effects on different Blm functions. Each deletion had distinct effects on different Blm activities. Deletion of either conserved region 1 (CR1) or conserved region 2 (CR2) compromised DSB repair through synthesis-dependent strand annealing and resulted in increased mitotic crossovers. In contrast, CR2 is critical for embryonic development but CR1 is not as important. CR1 deletion allows for proficient meiotic chromosome segregation but does lead to defects in meiotic crossover designation and patterning. Finally, deletion of CR2 does not lead to significant meiotic defects, indicating that while each region has overlapping functions, there are discreet roles facilitated by each. These results provide novel insights into functions of the N-terminal disordered region of Blm.

genetics↗

bioRxiv2023

Centromere-Proximal Suppression of Meiotic Crossovers in Drosophila is Robust to Changes in Centromere Number and Repetitive DNA Content

Accurate segregation of homologous chromosomes during meiosis depends on both the presence and regulated placement of crossovers (COs). The centromere effect (CE), or CO exclusion in pericentromeric regions of the chromosome, is a meiotic CO patterning phenomenon that helps prevent nondisjunction (NDJ), thereby protecting against chromosomal disorders and other meiotic defects. Despite being identified nearly a century ago, the mechanisms behind this fundamental cellular process remain unknown, with most studies of the Drosophila CE focusing on local influences of the centromere and pericentric heterochromatin. In this study, we sought to investigate whether dosage changes in centromere number and repetitive DNA content affect the strength of the CE, using phenotypic recombination mapping. Additionally, we also studied the effects of repetitive DNA function on CE strength using satellite-DNA binding protein mutants shown to have defective centromere clustering. Despite what previous studies suggest, our results show that the Drosophila CE is robust to dosage changes in centromere number and repetitive DNA content, and potentially also to repetitive DNA function. Our study suggests that the CE is unlikely to be spatially controlled, providing novel insight into the mechanisms behind the Drosophila centromere effect.

genetics↗

bioRxiv2022

The Effect of Repeat Length on Marcal1-Dependent Single-Strand Annealing in Drosophila

Proper repair of DNA double strand breaks (DSBs) is essential to maintenance of genomic stability and avoidance of genetic disease. Organisms have many ways of repairing DSBs, including use of homologous sequences through homology-directed repair (HDR). While HDR repair is often error-free, in single-strand annealing (SSA) homologous repeats flanking a DSB are annealed to one another, leading to deletion of one repeat and the intervening sequences. Studies in yeast have shown a relationship between the length of the repeat and SSA efficacy. We sought to determine the effects of homology length on SSA in Drosophila, as Drosophila uses a different annealing enzyme (Marcal1) than yeast. Using an in vivo SSA assay, we show that 50 base pairs (bp) is insufficient to promote SSA and that 500-2000 bp is required for maximum efficiency. Loss of Marcal1 generally followed the same homology length trend as wild-type flies, with SSA frequencies reduced to about a third of wild-type frequencies regardless of homology length. Interestingly, we find a difference in SSA rates between 500 bp homologies that align to the annealing target either nearer or further from the DSB, a phenomenon that may be explained by Marcal1 dynamics. This study gives insights into Marcal1 function and provides important information to guide design of genome engineering strategies that use SSA to integrate linear DNA constructs into a chromosomal DSB.

genetics↗

bioRxiv2021

Blm Helicase Facilitates Rapid Replication of Repetitive DNA Sequences in early Drosophila Development

The absence of functional BLM DNA helicase, a member of the RecQ family of helicases, is responsible for the rare human disorder Bloom Syndrome, which results in developmental abnormalities, DNA repair defects, genomic instability, and a predisposition to cancer. In Drosophila melanogaster, the orthologous Blm protein is essential during early development when the embryo is under the control of maternal gene products. We show that lack of functional maternal Blm during the syncytial cell cycles of Drosophila embryonic development results in severe nuclear defects and lethality. Amongst the small fraction of embryos from Blm mutant mothers that survive to adulthood, a prominent sex-bias favors the class that inherits less repetitive DNA content, which serves as an endogenous source of replication stress. This selection against repetitive DNA content reflects a role for Blm in facilitating replication through repetitive sequences during the rapid S-phases of syncytial cell cycles. During these syncytial cycles, Blm is not required for complex DNA double-strand break repair; however, the progeny sex-bias resulting from the absence of maternal Blm is exacerbated by repetitive DNA sequences and by the slowing of replication fork progression, suggesting that the essential role for Blm during this stage is to manage replication fork stress brought about by impediments to fork progression. Additionally, our data suggest that Blm is only required to manage this replication stress during embryonic development, and likely only during the early, rapid syncytial cell cycles, and not at later developmental stages. These results provide novel insights into Blm function throughout development.

genetics↗

bioRxiv2020

DNA polymerase theta suppresses mitotic crossing over

Polymerase theta-mediated end joining (TMEJ) is a chromosome break repair pathway that is able to rescue the lethality associated with the loss of proteins involved in early steps in homologous recombination (e.g., BRCA1/2). This is due to the ability of polymerase theta (Pol {theta}) to use resected, 3 single stranded DNA tails to repair chromosome breaks. These resected DNA tails are also the starting substrate for homologous recombination. However, it remains unknown if TMEJ can compensate for the loss of proteins involved in more downstream steps during homologous recombination. Here we expand the number of homologous recombination proteins synthetic lethal with Pol {theta} to the Holliday junction resolvases SLX4 and GEN1. SLX4 and GEN1 are required for viability in the absence of Pol {theta} in Drosophila melanogaster, and lack of all three proteins results in very high levels of apoptosis. We observe that flies deficient in Pol {theta} and SLX4 are extremely sensitive to DNA damaging agents, and mammalian cells require either Pol {theta} or SLX4 to survive. Our results suggest that TMEJ and Holliday junction formation/resolution share a common DNA substrate, likely a homologous recombination intermediate, that when left unrepaired leads to cell death. One major consequence of Holliday junction resolution by SLX4 and GEN1 is cancer-causing loss of heterozygosity due to mitotic crossing over. We measured mitotic crossovers in flies after a Cas9-induced chromosome break, and observed that this mutagenic form of repair is increased in the absence of Pol {theta}. This demonstrates that TMEJ can function upstream of the Holiday junction resolvases to protect cells from loss of heterozygosity. Our work argues that Pol {theta} can thus compensate for the loss of the Holliday junction resolvases by utilizing homologous recombination intermediates, suppressing mitotic crossing over and preserving the genomic stability of cells. Author summaryChromosome breaks are a common threat to the stability of DNA. Mutations in genes involved in the early steps of homologous recombination (BRCA1 and BRCA2), a mostly error-free chromosome break repair pathway, lead to hereditary breast cancer. Cells lacking BRCA1 and BRCA2 rely on DNA polymerase theta, a key protein for a more error-prone pathway, for survival. Using fruit flies and mammalian cells, we have shown that mutations in genes involved in later steps of homologous recombination (SLX4 and GEN1) also make cells reliant on polymerase theta. Moreover, we have shown that polymerase theta acts upstream of a type of homologous recombination that is error-prone and depends on SLX4 and GEN1. This form of homologous recombination, termed Holliday junction resolution, creates mitotic crossovers, which can lead to loss of heterozygosity and cancer. Our results expand the cellular contexts that make cells depend on polymerase theta for survival, and the substrates that this protein can use to repair chromosome breaks.

genetics↗

bioRxiv2020

Collaboration between homologous recombination and non-homologous end joining in repair of meiotic double-strand breaks in Drosophila

Programmed DNA double-strand breaks (DSBs) made during meiosis are repaired by recombination with the homologous chromosome to generate, at selected sites, reciprocal crossovers that are critical for the proper separation of homologs in the first meiotic divisions. Backup repair processes can compensate when the normal meiotic recombination processes are non-functional. We describe a novel backup repair mechanism that occurs when the homologous chromosome is not available in Drosophila melanogaster meiosis. In the presence of a previously described mutation (Mcm5A7) that disrupts chromosome pairing, DSB repair is initiated by homologous recombination but is completed by non-homologous end joining (NHEJ). Remarkably, this process yields precise repair products. Our results provide support for a recombination intermediate recently discovered in mouse meiosis, in which an oligonucleotide bound to the Spo11 protein that catalyzes DSB formation remains bound after resection. We propose that this oligonucleotide functions as a primer for fill-in synthesis to allow scarless repair by NHEJ.

genetics↗

bioRxiv2019

Mechanistic Basis for Microhomology Identification and Genome Scarring by Polymerase Theta

DNA Polymerase Theta mediates an end joining pathway (TMEJ) that repairs chromosome breaks. It requires resection of broken ends to generate long, 3 single stranded DNA tails, annealing of complementary sequence segments (microhomologies) in these tails, followed by microhomology-primed synthesis sufficient to resolve broken ends. The means by which microhomologies are identified is thus a critical step in this pathway, but is not understood. Here we show microhomologies are identified by a scanning mechanism initiated from the 3 terminus and favoring bi-directional progression into flanking DNA, typically to a maximum of 15 nucleotides into each flank. Polymerase theta is frequently insufficiently processive to complete repair of breaks in microhomology-poor, AT-rich regions. Aborted synthesis leads to one or more additional rounds of microhomology search, annealing, and synthesis; this promotes complete repair in part because earlier rounds of synthesis generate microhomologies de novo that are sufficiently long that synthesis is more processive. Aborted rounds of synthesis are evident in characteristic genomic scars as insertions of 3-30 bp of sequence that is identical to flanking DNA ("templated" insertions). Templated insertions are present at higher levels in breast cancer genomes from patients with germline BRCA1/2 mutations, consistent with an addiction to TMEJ in these cancers. Our work thus describes the mechanism for microhomology identification, and shows both how it mitigates limitations implicit in the microhomology requirement, and generates distinctive genomic scars associated with pathogenic genome instability.

molecular biology↗

bioRxiv2019

The Drosophila melanogaster ortholog of RFWD3 functions independently of RAD51 during DNA repair

Repair of damaged DNA is required for the viability of all organisms. Studies in Drosophila melanogaster, driven by the power of genetic screens, pioneered the discovery and characterization of many genes and pathways involved in DNA repair in animals. However, fewer than half of the alleles identified in these screens have been mapped to a specific gene, leaving a potential for new discoveries in this field. Here we show that the previously uncharacterized mutagen sensitive gene mus302 codes for the Drosophila melanogaster ortholog of the E3 ubiquitin ligase RING finger and WD domain protein 3 (RFWD3). In human cells, RFWD3 promotes ubiquitylation of RPA and RAD51 to facilitate repair of collapsed replication forks and double strand breaks through homologous recombination. Despite the high similarity in sequence to the human ortholog, our evidence fails to support a role for Mus302 in the repair of these types of damage. Last, we observe that the N-terminal third of RFWD3 is only present in mammals and absent in the rest of vertebrates and invertebrates. We propose that the additional N-terminal portion accounts for the acquisition of a new biological function in mammals that explains the functional differences between the human and the fly orthologs, and that Drosophila Mus302 may retain the ancestral function of the protein.

genetics↗

bioRxiv2019

Centromere-proximal meiotic crossovers in Drosophila melanogaster are suppressed by both highly-repetitive heterochromatin and the centromere effect

Crossovers are essential in meiosis of most organisms to ensure the proper segregation of chromosomes. The lack or improper placement of crossovers can result in nondisjunction and aneuploidy in progeny. Crossovers near the centromere can cause nondisjunction; centromere-proximal crossovers are suppressed by what is termed the centromere effect, but the mechanism is unknown. Here, we investigate contributions to centromere-proximal crossover suppression in Drosophila melanogaster. We mapped a large number of centromere-proximal crossovers and find that crossovers are essentially absent from the highly-repetitive (HR)-heterochromatin surrounding the centromere but occur at a low frequency within the less-repetitive (LR)-heterochromatic region and adjacent euchromatin. Previous research suggested that flies that lack the Bloom syndrome helicase (Blm) lose meiotic of crossover patterning, including the centromere effect. Mapping of centromere-proximal crossovers in Blm mutants reveals that the suppression within the HR-heterochromatin is intact, but the centromere effect is lost. We conclude that centromere-proximal crossovers are suppressed by two separable mechanisms: the HR-heterochromatin effect, which completely suppresses crossovers in the HR-heterochromatin, and the centromere effect, which suppresses crossovers with a dissipating effect with distance from the centromere.

genetics↗

bioRxiv2019

Centromere clustering stabilizes meiotic homolog pairing

During meiosis, each chromosome must selectively pair and synapse with its own unique homolog to enable crossover formation and subsequent segregation. How homolog pairing is maintained in early meiosis to ensure synapsis occurs exclusively between homologs is unknown. We aimed to further understand this process by utilizing a unique Drosophila meiotic mutant, Mcm5A7. We found that Mcm5A7 mutants are proficient in homolog pairing at meiotic onset yet fail to maintain pairing as meiotic synapsis ensues, causing seemingly-normal synapsis between non-homologous loci. This pairing defect corresponds with a reduction of SMC1-dependent centromere clustering at meiotic onset. Overexpressing SMC1 in this mutant significantly restores centromere clustering, homolog pairing, and crossover formation. These data indicate that the initial meiotic pairing of homologs is not sufficient to yield synapsis between exclusively between homologs and provide a model in which meiotic homolog pairing must be stabilized by SMC1-dependent centromere clustering to ensure proper synapsis.

cell biology↗

bioRxiv2018

Genetic analysis reveals novel roles for mei-MCMs during meiotic recombination in Drosophila

Crossover formation as a result of meiotic recombination is vital for proper segregation of homologous chromosomes at the end of meiosis I. In many organisms, crossovers are generated through two crossover pathways: Class I and Class II. To ensure accurate crossover formation, meiosis-specific protein complexes regulate the degree in which each pathway is used. One such complex is the mei-MCM complex, which contains MCM (mini-chromosome maintenance) and MCM-like proteins REC (ortholog of Mcm8), MEI-217, and MEI-218, collectively called the mei-MCM complex. The mei-MCM complex genetically promotes Class I crossovers and inhibits Class II crossovers in Drosophila, but it is unclear how individual mei-MCM proteins contribute to crossover regulation. In this study, we perform genetic analyses to understand how specific regions and motifs of mei-MCM proteins contribute to Class I and II crossover formation and distribution. Our analyses show that the long, disordered N-terminus of MEI-218 is dispensable for crossover formation, and that mutations that disrupt RECs Walker A and B motifs differentially affect Class I and Class II crossover formation. In Rec Walker A mutants, Class I crossovers exhibit no change, but Class II crossovers are increased. However, in rec Walker B mutants, Class I crossovers are severely impaired, and Class II crossovers are increased. These results suggest that REC may form multiple complexes that exhibit differential REC-dependent ATP binding and hydrolyzing requirements. These results provide genetic insight into the mechanisms through which mei-MCM proteins promote Class I crossovers and inhibit Class II crossovers.

genetics↗

bioRxiv2017

Meiotic Crossover Patterning In The Absence Of ATR: Loss Of Interference And Assurance But Not The Centromere Effect

Meiotic crossovers must be properly patterned to ensure accurate disjunction of homologous chromosomes during meiosis I. Disruption of the spatial distribution of crossovers can lead to nondisjunction, aneuploidy, gamete dysfunction, miscarriage, or birth defects. One of the earliest identified genes in involved proper crossover patterning is textitDrosophila mei-41, which encodes the ortholog of the checkpoint kinase ATR. Analysis of hypomorphic mutants suggested the existence of crossover patterning defects, but it was not possible to assess this in null mutants because of maternal-effect embryonic lethality. To overcome this lethality, we constructed mei-41 null mutants in which we expressed wild-type Mei-41 in the germline after completion of meiotic recombination, allowing progeny to survive. We find that crossovers are decreased to about one third of wild-type levels, but the reduction is not uniform, being less severe in the proximal regions of 2L than in medial or distal 2L or on the X chromosome. None of the crossovers formed in the absence of Mei-41 require Mei-9, the presumptive meiotic resolvase, suggesting that Mei-41 functions everywhere, despite the differential effects on crossover frequency. Interference appears to be significantly reduced or absent in mei-41 mutants, but the reduction in crossover density in centromere-proximal regions is largely intact. We propose that crossover patterning is achieved in a stepwise manner, with the crossover suppression related to proximity to the centromere occurring prior to and independently of crossover designation and enforcement of interference. In this model, Mei-41 has an essential after the centromere effect is established but before crossover designation and interference occur.

genetics↗