Biology subjects

Freudenreich, C. H.

Publications and source records attributed to Freudenreich, C. H..

7 recordsLinked to original sources

bioRxiv2026

A Novel ILP Framework to Identify Compensatory Pathways in Genetic Interaction Networks with GIDEON

In Bakers yeast, there exists a comprehensive collection of pairwise epistasis experiments that, for nearly every pair of non-essential genes, measures the growth of the double-knockout strain as compared to its component single knockouts. This data can be represented as a weighted signed graph termed the genetic interaction network, and we introduce a new ILP-based method named GIDEON to search for a diverse collection of Between-Pathway Models (BPMs) in this network, where BPMs are a graph motif signature that indicates potential compensatory pathways in the genetic interaction network. With both an improved distribution-informed edge weighting scheme and an improved ILP method, GIDEON produces BPM collections that are substantially larger and with better functional enrichment compared to previous methods. We find some interesting new BPM gene sets including one with potential insights into antifungal drug targets through ties between ergosterol and aromatic amino acid biosynthesis.

bioinformatics↗

bioRxiv2026

Cruciform-forming AT/TA repeats are acted upon by structure selective endonucleases and Rad51 prior to repositioning to the nuclear periphery for repair

Structure-forming DNA repeats can pose a barrier to DNA replication and repair, creating chromosomal fragile sites. An AT/TA DNA repeat, derived from the Flex1 region of human common fragile site FRA16D, can form hairpin and cruciform structures which interfere with DNA replication. When inserted into the S. cerevisiae genome, the Flex1(AT)34 repeat stimulates chromosome deletions in a manner dependent on the Mus81-Mms4 nuclease and the SLX4 nuclease scaffold. It was previously found that hairpin forming CAG/CTG repeats move to the nuclear periphery to maintain genomic stability. Here, we show that a structure forming AT/TA repeat also relocalizes to the nuclear periphery in late S/G2 phase in a replication and length-dependent manner. In contrast to the CAG repeat, this shift in nuclear positioning is dependent on polySUMOylation and the activity of the Mus81-Mms4 nuclease. Processing by the Mre11 nuclease and Rad51-dependent strand exchange occurs prior to repositioning. Replication analysis indicates that the replisome likely bypasses the AT/TA repeat leaving behind a DNA structure that initiates relocation to the nuclear periphery. We conclude that AT/TA repeats form post-replicative DNA structures that are targeted for nuclease cleavage and require hairpin processing and repositioning to the nuclear periphery for homologous recombination-dependent repair.

genetics↗

bioRxiv2024

C9orf72 expansion creates the unstable folate-sensitive fragile site FRA9A

The hyper-unstable Chr9p21 locus, harbouring the interferon gene cluster, oncogenes and C9orf72, is linked to multiple diseases. C9orf72 (GGGGCC)n expansions (C9orf72Exp) are associated with incompletely penetrant amyotrophic lateral sclerosis, frontotemporal dementia and autoimmune disorders. C9orf72Exp patients display hyperactive cGAS-STING-linked interferon immune and DNA damage responses, but the source of immuno-stimulatory or damaged DNA is unknown. Here, we show C9orf72Exp in pre-symptomatic and ALS-FTD patient cells and brains cause the folate- sensitive chromosomal fragile site, FRA9A. FRA9A centers on >33kb of C9orf72 as highly-compacted chromatin embedded in an 8.2Mb fragility zone spanning 9p21, encompassing 46 genes, making FRA9A one of the largest fragile sites. C9orf72Exp cells show chromosomal instability, heightened global- and Chr9p-enriched sister-chromatid exchanges, truncated-Chr9s, acentric-Chr9s and Chr9-containing micronuclei, providing endogenous sources of damaged and immunostimulatory DNA. Cells from one C9orf72Exp patient contained highly-rearranged FRA9A-expressing Chr9 with Chr9-wide dysregulated gene expression. Somatic C9orf72Exp repeat instability and chromosomal fragility are sensitive to folate-deficiency. Age-dependent repeat instability, chromosomal fragility, and chromosomal instability can be transferred to CNS and peripheral tissues of transgenic C9orf72Exp mice, implicating C9orf72Exp as the source. Our results highlight unappreciated effects of C9orf72 expansions that trigger vitamin-sensitive chromosome fragility, adding structural variations to the disease-enriched 9p21 locus, and likely elsewhere. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/620312v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@154b23borg.highwire.dtl.DTLVardef@1814a6dorg.highwire.dtl.DTLVardef@13d713eorg.highwire.dtl.DTLVardef@d5d994_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2024

The DNA Damage Checkpoint Targets the Kinetochore for Relocation of Collapsed Forks to the Periphery

Hairpin forming expanded CAG/CTG repeats pose significant challenges to DNA replication which can lead to replication fork collapse. Long CAG/CTG repeat tracts relocate to the nuclear pore complex to maintain their integrity. Forks impeded by DNA structures are known to activate the DNA damage checkpoint, thus we asked whether checkpoint proteins play a role in relocation of collapsed forks to the nuclear periphery in S. cerevisiae. We show that relocation of a (CAG/CTG)130 tract is dependent on activation of the Mrc1/Rad53 replication checkpoint. Further, checkpoint-mediated phosphorylation of the kinetochore protein Cep3 is required for relocation, implicating detachment of the centromere from the spindle pole body. Activation of this pathway leads to DNA damage-induced microtubule recruitment to the repeat. These data suggest a role for the DNA replication checkpoint in facilitating movement of collapsed replication forks to the nuclear periphery by centromere release and microtubule-directed motion. HighlightsO_LIThe DNA replication checkpoint initiates relocation of a structure-forming CAG repeat tract to the nuclear pore complex (NPC) C_LIO_LIThe importance of Mrc1 (hClaspin) implicates fork uncoupling as the initial checkpoint signal C_LIO_LIPhosphorylation of the Cep3 kinetochore protein by Dun1 kinase allows for centromere release, which is critical for collapsed fork repositioning C_LIO_LIDamage-inducible nuclear microtubules (DIMs) colocalize with the repeat locus and are required for relocation to the NPC C_LIO_LIEstablishes a new role for the DNA replication and DNA damage checkpoint response to trigger repositioning of collapsed forks within the nucleus. C_LI

cell biology↗

bioRxiv2022

Structure-forming CAG/CTG repeats interfere with gap repair to cause repeat expansions and chromosome breaks

Expanded CAG/CTG repeats are sites of DNA damage, leading to changes in repeat length. To determine how ssDNA gap filling affects repeat instability, we inserted (CAG)70 or (CTG)70 repeats into a single-strand annealing (SSA) assay system such that resection and filling in the ssDNA gap would occur across the repeat tract. After resection, when the CTG sequence was the single-stranded template for fill-in synthesis, repeat contractions were elevated and the ssDNA created a fragile site that led to large deletions involving flanking homologous sequences. In contrast, resection was inhibited when CTG was on the resected strand, resulting in repeat expansions. Deleting Rad9, the ortholog of 53BP1, rescued repeat instability and lost viability by increasing resection and fill-in speed. Deletion of Rad51 increased CTG contractions and decreased survival, implicating Rad51 in protecting ssDNA during gap filling. Taken together, DNA sequence within a single-stranded gap determines repair kinetics, fragility, and repeat instability.

genetics↗

bioRxiv2021

THO and TRAMP complexes prevent transcription-replication conflicts, DNA breaks, and CAG repeat contractions

Expansion of structure-forming CAG/CTG repetitive sequences is the cause of several neurodegenerative disorders and deletion of repeats is a potential therapeutic strategy. Transcription-associated mechanisms are known to cause CAG repeat instability. In this study, we discovered that Thp2, an RNA export factor and member of the THO complex, and Trf4, a key component of the TRAMP complex involved in nuclear RNA degradation, are necessary to prevent CAG fragility and repeat contractions in a S. cerevisiae model system. Depletion of both Thp2 and Trf4 proteins causes a highly synergistic increase in CAG repeat fragility, indicating a complementary role of the THO and TRAMP complexes in preventing genome instability. Loss of either Thp2 or Trf4 causes an increase in RNA polymerase stalling at the CAG repeats and genome-wide transcription-replication conflicts (TRCs), implicating impairment of transcription elongation as a cause of CAG fragility and instability in their absence. Analysis of the effect of RNase H1 overexpression on CAG fragility and TRCs suggests that co-transcriptional R-loops are the main cause of CAG fragility in the thp2{Delta} mutants. In contrast, CAG fragility and TRCs in the trf4{Delta} mutant can be compensated for by RPA overexpression, suggesting that excess unprocessed RNA in TRAMP4 mutants leads to reduced RPA availability and high levels of TRCs. Our results show the importance of RNA surveillance pathways in preventing RNAPII stalling, TRCs, and DNA breaks, and show that RNA export and RNA decay factors work collaboratively to maintain genome stability.

genetics↗

bioRxiv2018

Saccharomyces cerevisiae H2A copies differentially contribute to recombination and CAG/CTG repeat maintenance, with a role for H2A.1 threonine 126

DNA are sites of genomic instability. Long CAG/CTG repeats form hairpin structures, are fragile, and can expand during DNA repair. The chromatin response to DNA damage can influence repair fidelity, but the knowledge of chromatin modifications involved in maintaining repair fidelity within repetitive DNA is limited. In a screen for CAG repeat fragility in Saccharomyces cerevisiae, histone 2A copy 1 (H2A.1) was identified to protect the repeat from increased rates of breakage. To address the role of H2A in CAG repeat instability, we tested the effect of deleting each histone H2 subytpe. Whereas deletion of HTA2, HTZ1, HTB1, and HTB2 did not significantly affect CAG repeat maintenance, deletion of HTA1 resulted in increased expansion frequency. Notably, mutation of threonine 126, unique to H2A.1, to a non-phosphorylatable alanine increased CAG repeat instability to a similar level as the hta1{Delta} mutant. CAG instability in the absence of HTA1 or mutation to hta1-T126A was dependent on the presence of the homologous recombination (HR) repair proteins Rad51, Rad52, and Rad57, and the Pol{delta} subunit Pol32. In addition, sister chromatid recombination (SCR) was suppressed in the hta1{Delta} and hta1-T126A mutants and this suppression was epistatic to pol32{Delta}. Finally, break-induced replication (BIR) is impaired in the hta1{Delta} mutant, resulting in an altered repair profile. These data reveal differential roles for the H2A subtypes in DNA repair and implicate a new role for H2A.1 threonine-126 phosphorylation in mediating fidelity during HR repair and promoting SCR. Using a fragile, repetive DNA element to model endogenous DNA damage, our results demonstrate that H2A.1 plays a greater role than H2A.2 in promoting homology-dependent repair, suggesting H2A.1 is the true homolog of mammalian H2AX, whereas H2A.2 is functionally equivalent to mammalian H2A.\n\nAuthor SummaryCAG/CTG trinuncleotide repeats are fragile sequences that when expanded can cause human disease. To evaluate the role of S. cerevisiae histone H2A copies in DNA repair, we have measured instability of an expanded CAG/CTG repeat tract and repair outcomes in H2A mutants. Although the two copies of H2A are nearly identical in amino acid sequence, we found that the CAG repeat is more unstable in the absence of H2A copy 1 (H2A.1) than H2A copy 2, and that this role appears to be partially dependent on a phosphorylatable threonine at residue 126 in the C-terminal tail of H2A.1. Further, we show through a series of genetic assays that H2A.1 plays a role in promoting homologous recombination events, including sister chromatid recombination and break-induced replication. Our results uncover a role for H2A.1 in mediating fidelity of repair within repetitive DNA, and demonstrate that modification of its unique Thr126 residue plays a role in regulating SCR. Given the dependence of HR repair on H2A.1 but not H2A.2, we conclude that H2A.1 plays a greater repair-specific role in the cell and therefore would be the true homolog of mammalian H2AX.

genetics↗