Biology subjects

Raghuraman, M. K.

Publications and source records attributed to Raghuraman, M. K..

9 recordsLinked to original sources

bioRxiv2026

Making sense of chaos: replication and segregation defects in the yeast mcm4Chaos3 mutant

Genome instability is a defining feature of cancer and is strongly influenced by alterations in the fidelity and timing of DNA replication. The conserved Mini-Chromosome Maintenance (MCM 2-7) helicase is a central component of the eukaryotic DNA replication machinery, playing critical roles in origin licensing and activation. Genetic screens for genome instability mutations in mice identified mcm4Chaos3, a hypomorphic allele of MCM4 associated with the formation of micronuclei and tumorigenesis, suggesting that the mcm4Chaos3 allele causes genome instability. To define the mechanistic basis of this instability, we examined the effects of the corresponding mutation in Saccharomyces cerevisiae. We found that mcm4Chaos3 selectively compromises origin firing at a subset of early S-phase origins, with pronounced effects at many centromere-proximal origins. Chromosome V, containing the sensitive centromere-adjacent ARS510, exhibited elevated loss rates that were rescued by replacement with the robust early-firing origin ARS305, restoring both origin activity and chromosome stability. Together these findings demonstrate that mcm4Chaos3 compromises genome stability by impairing a subset of early, centromere-proximal origins that are intrinsically sensitive to origin activation capacity. Our results establish that robust origin function near centromeres is critical for accurate chromosome segregation and reveal how subtle perturbations in helicase function can lead to chromosome instability.

genetics↗

bioRxiv2026

Beyond ERCs: exploring catastrophic forms of rDNA instability in aging yeast

Single-celled organisms grown in identical conditions have variable life spans. Identifying the factors that drive the inherent variability in life span is crucial for our understanding of aging at a fundamental level. Here, we revisit the role of chromosome XII instability as a source of life span variability in aging populations of the budding yeast, Saccharomyces cerevisiae. We followed populations of mother cells as they aged and quantified changes in karyotype, DNA content, and aberrant DNA structures, including the production of extrachromosomal rDNA circles (ERCs). We found that cells massively amplified their rDNA both as ERCs and as a structural form that could not be resolved on CHEF gels. We propose a model describing how these unresolved structures are generated. Our model, which we call CICR (Catastrophic IntraChromosomal Recombination), describes the consequences of recombination between repeats of different replication status. At the completion of replication, when all other replication forks have successfully terminated, CICR events leave behind a single, unopposed replication fork in a branched form of Chr XII that has profound consequences during mitosis and/or subsequent cycles. This form of instability within the ribosomal DNA can lead to a myriad of toxic recombination products that may contribute to the life span variability in isogenic populations of aging yeast. AUTHOR SUMMARYAging is an uphill battle against entropy. The wear and tear on biological processes in cells require surveillance mechanisms and repair processes to stave off the inevitable. A cells genome is constantly under external and internal insults that can ultimately lead to death. Known events that impact a cells DNA are damage from free radicals (produced by the very oxygen needed for survival), shortening of the caps on chromosomes (called telomeres) every time a cell divides, and mutations that accumulate over time that affect essential cell processes. Yeast is an excellent tool to examine specific causes of aging because of its asymmetric mode of cell division: a mother cell can divide a limited, but variable number of times before senescing. We have isolated cohorts of mother cells, examined their genomes over the course of their life span and find a new intrinsic insult that the genome must endure. We find, in addition to the accumulation of extrachromosomal copies of the genes that encode the RNA portion of ribosomes (rDNA), that cells accumulate a toxic product of genetic recombination within the rDNA. The chromosome that contains the rDNA becomes branched and/or fragmented which leads to cell death.

genetics↗

bioRxiv2026

Telomeric amplicons of SUL1 and Y' in yeast are generated by microhomology-mediated break induced replication occurring in cis

Gene amplification is a potent driver of evolution and is thought to contribute to genetic diseases, including cancer. The yeast Saccharomyces cerevisiae is a powerful organism for understanding amplification mechanisms. When yeast is grown long term in sulfate-limiting chemostats, amplification of the gene that encodes the primary sulfate transporter, SUL1, is a common outcome. Here we describe a form of SUL1 amplification in which multiple copies of the right terminal region of chromosome II are appended in tandem to a native telomere. We find this form of amplicon when we delete the origin of replication next to SUL1 or delete a variety of genes involved in DNA metabolism. It is the only form of amplification found in a yku70{Delta} mutant suggesting that unprotected telomeres are involved. We propose that these terminal addition events occur when the unprotected 3 G1-3T telomeric sequence invades a short ([~]7 bp) internal telomere sequence (ITS) to begin a form of microhomology-mediated break-induced replication (mmBIR) that has been documented in type-I survivors of telomerase mutants. In addition to amplification of the right end of chromosome II we also find that telomeres containing the sub-telomeric repeat Y experience similar tandem amplification events and show that their formation is reduced in a pol32{Delta} mutant, a gene required for mmBIR. Within individual amplicons the ITSs and Ys are nearly identical, suggesting that the multiple copies of the amplified region are generated in a single mmBIR event that we describe as pseudo-rolling circle mmBIR. A similar amplification event at the P-telomere of human chromosome 18 has four copies of a [~]54 kb region separated by ITSs of nearly identical size. This finding suggests that these additional copies of the terminal fragment of human chromosome 18 arose by the same pseudo-rolling circle mechanism, perhaps during a period of telomeric stress. AUTHOR SUMMARYThe human genome is peppered with duplicates (or higher numbers) of segments that are located at sites both nearby and distant from the original, ancestral segments. These Copy Number Variants, or CNVs, appear to be highly variable among different individuals and are being examined with great interest as potential loci associated with genetic disease. Experimentally determining how these CNVs arise and become distributed across the genome is nearly impossible using humans. We are using budding yeast as the model organism to explore mechanisms of gene amplification. In this work we show that by destabilizing the ends of yeast chromosomes (telomeres) or by interfering with genes involved in the replication, repair, or recombination of DNA results in a specific form of segmental copy number increase that is initiated at telomeres. We propose that a telomere invades an internal chromosome site and sets up a pseudo-circular template for conservative DNA replication. The outcome is a chromosome with multiple, identical copies of a chromosome end arranged in tandem. We believe that it is also a major mechanism used by cells to repair telomeres that have become eroded during aging.

genetics↗

bioRxiv2024

rDNA copy number variation in yeast alters response to environmental conditions

Ribosomal DNA (rDNA) in eukaryotes is maintained in hundreds of copies with rDNA copy number varying greatly among individuals within a species. In the budding yeast Saccharomyces cerevisiae, the rDNA copy number across wild isolates ranges from 90 to 300 copies. Previous studies showed that 35 rDNA copies are sufficient for ribosome biogenesis in this yeast and enable wild-type-like growth in standard laboratory growth conditions. We addressed two major questions concerning rDNA copy number variation in this yeast: (1) What are the fitness consequences of rDNA copy number variation outside and within the natural range in standard laboratory growth conditions? (2) Do these fitness effects change in different growth conditions? We used growth competitions to compare the fitness effects of rDNA copy number variation in otherwise isogenic strains whose rDNA copy number ranged from 35 to 200. In standard growth conditions, we found that fitness gradually increases from 35 rDNA copies until reaching a plateau that spans from 98 to 160 rDNA copies, well within the natural range. However, rDNA copy number-dependent fitness differed across environments. Compared to standard growth conditions, strains with higher rDNA copy numbers showed improved fitness when grown at increased temperature or in glycerol media. Moreover, the gradual fitness increase with increasing rDNA copy number in standard growth conditions gave way to a markedly lower fitness of strains with copy numbers below the natural range in these two stress conditions. These results suggest that selective pressures drive rDNA copy number in this yeast to at least ~100 copies and that a higher number of copies buffers against environmental stress. The similarity of the S. cerevisiae rDNA copy number range to the ranges reported in C. elegans, D. melanogaster, and humans points to conserved selective pressures maintaining the range of natural rDNA copy number in these highly diverse species.

genetics↗

bioRxiv2023

A unifying model that explains the origins of human inverted copy number variants

With the release of the telomere-to-telomere human genome sequence and the availability of both long-read sequencing and optical genome mapping techniques, the identification of copy number variants and other structural variants is providing new insights into human genetic disease. Different mechanisms have been proposed to account for the novel junctions in these complex architectures, including aberrant forms of DNA replication, non-allelic homologous recombination and various pathways that repair DNA breaks. Here we have focused on a set of structural variants that include an inverted segment and propose that they share a common initiating event: an inverted triplication with long, unstable palindromic junctions. The secondary rearrangement of these palindromes gives rise to the various forms of inverted structural variants. We postulate that this same mechanism (ODIRA: Origin Dependent Inverted Repeat Amplification) that creates the inverted copy number variants in inherited syndromes also generates the palindromes found in cancers.

genetics↗

bioRxiv2023

Template switching between the leading and lagging strands at replication forks generates inverted copy number variants through hairpin-capped extrachromosomal DNA

Inherited and de novo copy number variants (CNVs) are increasingly found to be correlated with human developmental and cancerous phenotypes. Several models for template switching during replication have been proposed to explain the generation of these gross chromosomal rearrangements. We proposed a model of template switching (ODIRA--origin dependent inverted repeat amplification) in which simultaneous ligation of the leading and lagging strands at diverging replication forks could generate segmental inverted triplications through an extrachromosomal inverted circular intermediate. Here, we created a genetic assay using split-ura3 cassettes to trap the proposed inverted intermediate. However, instead of recovering circular inverted intermediates, we found inverted linear chromosomal fragments ending in native telomeres--suggesting that a template switch had occurred at the centromere-proximal fork of a replication bubble. As telomeric inverted hairpin fragments can also be created through double strand breaks we tested whether replication errors or repair of double stranded DNA breaks were the most likely initiating event. The results from CRISPR/Cas9 cleavage experiments and growth in the replication inhibitor hydroxyurea indicate that it is a replication error, not a double stranded break that creates the inverted junctions. Since inverted amplicons of the SUL1 gene occur during long-term growth in sulfate-limited chemostats, we sequenced evolved populations to look for evidence of linear intermediates formed by an error in replication. All of the data are compatible with a two-step version of the ODIRA model in which sequential template switching at short inverted repeats between the leading and lagging strands at replication forks generates inverted interstitial triplications. AUTHOR SUMMARYChromosomal rearrangements are a potent source of genetic variation in humans and other organisms. One specific type of rearrangement involves the increase in copies of segments of the genome. The variation in gene dosage that these rearrangements can cause has been associated with a wide range of neurological and other human disorders. A specific puzzling form of copy number increase consists of three tandem copies with the central copy in inverted orientation. How this rearrangement occurs is of great interest, yet the mechanisms responsible are only inferred by examining the sequence of final inverted products. Yeast provides a unique model system to explore the underlying molecular defects that give rise to inverted triplications. While the favored hypothesis suggests that double stranded DNA repair is the causative agent, we find that a particular form of template switching between strands at the replication fork, not a double stranded DNA break, is the initiating event. Using the awesome power of yeast genetics, we provide evidence in two different assays for this unique replication error that we call ODIRA (for Origin Dependent Inverted Repeat Amplification) and propose that it can also explain this form of copy number variant seen in human evolution and disease.

genomics↗

bioRxiv2022

Hotspot of de novo telomere addition stabilizes linear amplicons in yeast grown in sulfate-limiting conditions

Evolution is driven by the accumulation of competing mutations that influence survival. A broad form of genetic variation is the amplification or deletion of DNA ([≥]50 bp) referred to as copy number variation. In humans, copy number variation may be inconsequential, contribute to minor phenotypic differences, or cause conditions such as birth defects, neurodevelopmental disorders, and cancers. To identify mechanisms that drive copy number variation, we monitored the experimental evolution of Saccharomyces cerevisiae populations grown under sulfate-limiting conditions. Cells with increased copy number of the gene SUL1, which encodes a primary sulfate transporter, exhibit a fitness advantage. Previously, we reported interstitial inverted triplications of SUL1 as the dominant rearrangement in a haploid population. Here, in a diploid population, we find instead that small linear fragments containing SUL1 form and are sustained over several generations. Many of the linear fragments are stabilized by de novo telomere addition within a telomere-like sequence near SUL1 (within the SNF5 gene). Using an assay that monitors telomerase action following an induced chromosome break, we show that this region acts as a hotspot of de novo telomere addition and that required sequences map to a region of <250 base pairs. Consistent with previous work showing that association of the telomere-binding protein Cdc13 with internal sequences stimulates telomerase recruitment, mutation of a four-nucleotide motif predicted to associate with Cdc13 abolishes de novo telomere addition. Our study suggests that internal telomere-like sequences that stimulate de novo telomere addition can contribute to adaptation by promoting genomic plasticity.

genetics↗

bioRxiv2021

A minimal rDNA array replicates early, delays genome replication, and uncouples anaphase entry from S phase completion

Eukaryotes maintain hundreds of copies of ribosomal DNA (rDNA), many more than required for ribosome biogenesis, suggesting a yet undefined role for large rDNA arrays outside of ribosomal RNA synthesis. We demonstrate that reducing the Saccharomyces cerevisiae rDNA array to 35 copies, which is sufficient for ribosome function, shifts rDNA from being the latest replicating region in the genome to one of the earliest. This change in replication timing results in delayed genome-wide replication and classic replication defects. We present evidence that the requirement for rDNA to replicate late, which is conserved among eukaryotes, also coordinates the completion of genome replication with anaphase entry through the proper sequestration of the mitotic exit regulator Cdc14p in the rDNA-containing nucleolus. Our findings suggest that, instead of being a passive repetitive element, the large late-replicating rDNA array plays an active role in genome replication and cell cycle control.

molecular biology↗

bioRxiv2018

The limiting DNA replication initiation factors Sld2 and Sld3 influence origin efficiency independent of origin firing time

Chromosome replication in Saccharomyces cerevisiae is initiated from roughly 300 origins that are regulated both by DNA sequence and by the limited abundance of four trans-acting initiation proteins (Sld2, Sld3, Dpb11 and Dbf4, collectively called \"SSDD\"). We set out to determine how the association of Sld2 or Sld3 at origins contributes to time of origin activation and/or origin efficiency using auxin-induced protein degradation to further decrease their abundance. Depleting cells of either factor slows growth rate, increases S-phase duration, and causes viability defects, without activating the S phase checkpoint. Chr XII is uniquely unstable with breakage occurring specifically within the rDNA locus. The efficiency of the rDNA origin is decreased while the onset of replication initiation is unchanged. We found that origin efficiency is reduced uniformly across the unique portions of the yeast genome. We conclude that the abundance of Sld2 and Sld3 contribute primarily to origin efficiency.

molecular biology↗