Biology subjects

Mattarocci, S.

Publications and source records attributed to Mattarocci, S..

10 recordsLinked to original sources

bioRxiv2026

Structural basis for continuous DNA-end protection during ligation of double-strand breaks in yeast Non-Homologous End-Joining

Non-homologous end joining (NHEJ) repairs DNA double-strand breaks by synapsing and ligating DNA ends. In vertebrates, DNA-PKcs promotes end alignment and processing, yet several eukaryotes - including budding yeast - perform NHEJ without DNA-PKcs, and the underlying mechanism remains unclear. Here we report cryo-electron microscopy structures of reconstituted Saccharomyces cerevisiae NHEJ synaptic complexes assembled on DNA ends bearing either 4-bp terminal microhomologies or blunt termini. On the microhomology substrate with a single 5-P, we capture a ligation-competent short-range complex in which a single Dnl4 catalytic core is fully ordered and engaged at the 5'-adenylated nick. When two 5-P are present, we resolve two predominant, coexisting DNA-aligned protective states in which both Dnl4 DBD-NTD modules occupy the break region with reciprocal geometries, supporting an alternating engagement model for sequential sealing of the two strands while maintaining continuous end association. In contrast, blunt-ended substrates yield a non-aligned protective configuration in which two Dnl4 catalytic modules constrain the DNA ends [~]30 [A] apart in a ligation-incompatible arrangement, providing a structural explanation for slow blunt-end joining in yeast. Together, these structures define architectural and mechanistic principles of DNA-PKcs-independent NHEJ.

molecular biology↗

bioRxiv2025

The shortest telomere in telomerase-negative cells triggers replicative senescence at a critical threshold length and also fuels genomic instability

In the absence of telomerase, telomere shortening triggers replicative senescence, a tumor suppressor mechanism that is also associated with oncogenic genomic instability. Yet, the precise mechanism that connects these seemingly opposing forces remains poorly understood. To directly study the complex interplay between senescence, telomere dynamics, and genomic instability, we developed a system in Saccharomyces cerevisiae to generate and track telomeres of precise length in the absence of telomerase. Using single-telomere and single-cell analyses combined with mathematical modeling, we identify a threshold length at which telomeres switch into dysfunction. A single shortest telomere below the threshold length is necessary and sufficient to trigger the onset of replicative senescence in a majority of cells. At population level, fluctuation assays establish that rare genomic instability arises predominantly in cis to the shortest telomere as Pol32-dependent non-reciprocal translocations that result in re-elongation of the shortest telomere and likely transient escape from senescence. The switch of the shortest telomere into dysfunction and subsequent processing in telomerase-negative cells thus serves as the mechanistic link between replicative senescence onset, genomic instability and the initiation of post-senescence survival.

molecular biology↗

bioRxiv2025

Rad5 and Telomere Replication

The DNA Damage Tolerance pathway (DDT) is one of the major mechanisms for resolving replication fork blocks. A central factor for DDT is the fork-associated clamp, PCNA, which can undergo to mono- or polyubiquitination, leading to error-free or error-prone modes of repair, respectively. The Rad5 protein in the yeast Saccharomyces cerevisiae plays important roles in both pathways: promoting the error-free mode by PCNA polyubiquitination and interacting with specialized DNA polymerases involved in the error-prone pathway. Rad5 also associates with telomeres, the repetitive DNA regions present at the ends of chromosomes. Telomeric DNA, tightly bound by tandem proteins arrays, poses unique challenges to replication fork progression. Here, I review the current understanding of the link between Rad5 and telomeres and present evidence that Rad5 is associated with yeast telomeres, with notable enrichment during telomere replication. This data highlights a connection between telomeres and a key DDT factor in unperturbed wild-type cells, raising intriguing possibilities about the functional interplay between telomere replication and DNA damage tolerance mechanisms.

molecular biology↗

bioRxiv2024

Restriction on Ku's Inward Translocation Caps Telomere Ends

Safeguarding chromosome ends against fusions via nonhomologous end joining (NHEJ) is essential for preserving genome integrity. Paradoxically, the conserved NHEJ core factor Ku binds telomere ends. How it is prevented from promoting NHEJ remains unclear, as does the mechanism that allows Ku to coexist with telomere-protective DNA binding proteins, e.g., Rap1 in Saccharomyces cerevisiae. Here, we reveal a direct role for Rap1 in the inhibition of Kus NHEJ function at telomeres. A single Rap1 molecule bound near a DNA end inhibits NHEJ in vivo without disrupting Ku presence. Consistent with this, Rap1 and Ku form a complex on short DNA duplexes in vitro. Cryo-EM and molecular modelling analysis of this complex shows that Rap1 obstructs Kus inward translocation on DNA - an essential step for NHEJ at broken ends. Nanopore sequencing of telomere fusions confirms the importance of this pathway in protecting native telomere ends. Collectively, our findings uncover a mechanism of telomere end protection mediated by restricting Kus inward translocation, a functional switch that prevents promiscuous NHEJ repair at telomeres.

molecular biology↗

bioRxiv2023

Telomere protein arrays stall DNA loop extrusion by condensin

DNA loop extrusion by SMC proteins is a key process underlying chromosomal organization. It is unknown how loop extruders interact with telomeres where chromosome ends are covered with a dense array of tens of neighboring DNA-binding proteins. Using complementary in vivo and in vitro single-molecule approaches, we study the interaction between loop-extruding condensin and arrays of Rap1, the double-stranded-DNA-binding telomeric protein of Saccharomyces cerevisiae. We show that dense linear Rap1 arrays can completely halt DNA loop extrusion, where the blocking efficiency depends on the array length and the DNA gap size between neighboring proteins. In cells, Rap1 arrays in the chromosome are found to act as contact insulators and to accumulate condensin at their borders, with direct implications for the resolution of dicentric chromosomes produced by telomere fusions. Our findings show that linear arrays of DNA-bound proteins can efficiently halt DNA loop extrusion by SMC proteins, which may impact a wide range of cellular processes from telomere functions to transcription and DNA repair.

molecular biology↗

bioRxiv2021

ChEC-seq: a robust method to identify protein-DNA interactions genome-wide

Mittal et al. (2021; first brought to our attention in May 2019) have raised concerns regarding the Chromatin Endogenous Cleavage-sequencing (ChEC-seq) technique (Zentner et al., 2015) that may create a false impression that this method has fundamental flaws which prevent one from distinguishing between signal and noise. Although Mittal et al. focus on studies of the global co-activators SAGA, TFIID and Mediator that we were not involved in, we feel obliged to highlight here several of our own publications (Albert et al., 2019; Bruzzone et al., 2018; Hafner et al., 2018; Kubik et al., 2019; Kubik et al., 2018), as well as recent unpublished data, that employed ChEC-seq and directly addressed the observation raised by Mittal et al. that cleavage maps for various MNase fusion proteins often qualitatively resemble each other and those generated by "free" (unfused) MNase. Our studies lay out a clear path for determining sites of preferential factor localization by normalization of ChEC-seq experimental data to matched free-MNase controls. They also demonstrate the use of in vivo functional assays to assess ChEC-seq reliability and reveal examples where ChEC-seq identifies functional binding sites missed by conventional ChIP-seq analysis.

molecular biology↗

bioRxiv2020

Maturation of telomere 3'-overhangs is linked to the replication stress response

Withdrawal StatementThe authors have withdrawn their manuscript because, upon reanalyzing the primary data it appears that the methods used by one of the authors, S.M., might have compromised the reliability of the data presented in Figure 1. We sincerely apologize for any harm or confusion this may cause to BioRxiv and its users.

molecular biology↗

bioRxiv2019

Opposing chromatin remodelers control transcription initiation frequency and start site selection

Precise nucleosome organization at eukaryotic promoters is thought to be generated by multiple chromatin remodeler (CR) enzymes and to affect transcription initiation. Using an integrated analysis of chromatin remodeler binding and nucleosome displacement activity following rapid remodeler depletion, we investigate the interplay between these enzymes and their impact on transcription in budding yeast. We show that many promoters are acted upon by multiple CRs that operate either cooperatively or in opposition to position the key transcription start site-associated +1 nucleosome. Functional assays suggest that +1 nucleosome positioning often reflects a trade-off between maximizing RNA Polymerase II recruitment and minimizing transcription initiation at incorrect sites. Finally, we show that nucleosome movement following CR inactivation usually results from the activity of another CR and that in the absence of any remodeling activity +1 nucleosomes maintain their positions. Our results provide a detailed picture of fundamental mechanisms linking promoter nucleosome architecture to transcription initiation.

molecular biology↗

bioRxiv2018

Sfp1 regulates transcriptional networks driving cell growth and division through multiple promoter binding modes

Understanding how transcriptional programs help to coordinate cell growth and division is an important unresolved problem. Here we report that the nutrient- and stress-regulated transcription factor Sfp1 is rate-limiting for expression of several large classes of genes involved in yeast cell growth, including ribosomal protein, ribosome biogenesis, and snoRNA genes. Remarkably, the spectrum of Sfp1 transcription effects is concordant with a combination of chromatin immunoprecipitation and chromatin endogenous cleavage binding analyses, which together provide evidence for two distinct modes of Sfp1 promoter binding, one requiring a co-factor and the other a specific DNA-recognition motif. In addition to growth-related genes, Sfp1 binds to and regulates the promoters of cell cycle \"START\" regulon genes, including the key G1/S cyclins CLN1 and CLN2. Our findings suggest that Sfp1 acts as a master regulator of cell growth and cell size by coordinating the expression of genes implicated in mass accumulation and cell division.

molecular biology↗

bioRxiv2018

Sequence-directed action of RSC remodeler and pioneer factors positions +1 nucleosome to facilitate transcription

Accessible chromatin is important for RNA polymerase II recruitment and transcription initiation at eukaryotic promoters. We investigated the mechanistic links between promoter DNA sequence, nucleosome positioning and transcription. Our results indicate that precise positioning of the transcription start site-associated +1 nucleosome in yeast is critical for efficient TBP binding, and is driven by two key factors, the essential chromatin remodeler RSC and a small set of ubiquitous pioneer transcription factors. We find no evidence for recruitment of RSC by pioneer factors, but show instead that the strength and directionality of RSC action on nucleosomes depends upon the arrangement of two specific DNA motifs that promote its binding and nucleosome displacement activity at promoters. Thus, despite their widespread co-localization, RSC and pioneer factors predominantly act independently to generate accessible chromatin. Our results provide insight into how promoter DNA sequence instructs trans-acting factors to control nucleosome architecture and stimulate transcription initiation.

molecular biology↗