Biology subjects

Alvino, G. M.

Publications and source records attributed to Alvino, G. M..

8 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↗

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↗

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↗

bioRxiv2021

Exceptional origin activation revealed by comparative analysis in two laboratory yeast strains

We performed a comparative analysis of replication origin activation by genome-wide single-stranded DNA mapping in two common laboratory strains of Saccharomyces cerevisiae challenged by hydroxyurea (HU), an inhibitor of the ribonucleotide reductase. By doing so we gained understanding of the impact on origin activation by three factors: replication checkpoint control, DNA sequence polymorphisms, and relative positioning of origin and transcription unit. Our analysis recapitulated the previous finding that the majority of origins are subject to checkpoint control by the Rad53 kinase when cells were treated with HU. In addition, origins either subject to Rad53 checkpoint control or impervious to it are largely concordant between the two strains. However, these two strains also produced different dynamics of origin activation. First, the W303-RAD53 cells showed a significant reduction of fork progression than A364a-RAD53 cells. This phenotype was accompanied by an elevated level of Rad53 phosphorylation in W303-RAD53 cells. Second, W303-rad53K227A checkpoint-deficient cells activated a greater number of origins accompanied by global reduction of ssDNA across all origins compared to A364a-rad53K227A cells; and this is correlated with lower expression level of the mutant protein in W303 than in A364a. We also show that sequence polymorphism in the consensus motifs of the replication origins plays a minor role in determining origin usage. Remarkably, eight strain-specific origins lack the canonical 11-bp consensus motif for autonomously replicating sequences in either strain background. Finally, we identified a new class of origins that are only active in checkpoint-proficient cells, which we named "Rad53-dependent origins". The only discernible feature of these origins is that they tend to overlap with an open reading frame, suggesting previously unexplored connection between transcription and origin activation. Our study presents a comprehensive list of origin usage in two diverse yeast genetic backgrounds, fine-tunes the different categories of origins with respect to checkpoint control, and provokes further exploration of the interplay between origin activation and transcription. Author SummaryComparative analysis of origins of replication in two laboratory yeast strains reveals new insights into origin activation, regulation and dependency on the Rad53 checkpoint kinase.

genomics↗

bioRxiv2019

Phenotypic and genotypic consequences of CRISPR/Cas9 editing of the replication origins in the rDNA of Saccharomyces cerevisiae

The complex structure and repetitive nature of eukaryotic ribosomal DNA (rDNA) is a challenge for genome assembly, and thus, the consequences of sequence variation in rDNA remain unexplored. However, renewed interest in the role that rDNA variation may play in diverse cellular functions, aside from ribosome production, highlights the need for a method that would permit genetic manipulation of the rDNA. Here, we describe a CRISPR/Cas9 based strategy to edit the rDNA locus in the budding yeast Saccharomyces cerevisiae. Using this approach, we modified the endogenous rDNA origin of replication in each repeat by deleting or replacing its consensus sequence. We characterized the transformants that have successfully modified their rDNA locus and propose a mechanism for how CRISPR/Cas9 mediated editing of the rDNA occurs. In addition, we carried out extended growth and life span experiments to investigate the long-term consequences that altering the rDNA origin of replication has on cellular health. We find that long-term growth of the edited clones results in faster growing suppressors that have acquired segmental aneusomy of the rDNA containing region of chr XII or aneuploidy of chromosomes XII, II, or IV. Furthermore, we find that all edited isolates suffer a reduced life span, irrespective of their levels of extrachromosomal rDNA circles. Our work demonstrates that it is possible to quickly, efficiently and homogeneously edit the rDNA locus via CRISPR/Cas9. It serves as a model for modifying other parts of the rDNA and, more generally, for editing other tandemly repeated sequences in higher eukaryotes.

genetics↗

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↗