Biology subjects

BUSSO, D.

Publications and source records attributed to BUSSO, D..

4 recordsLinked to original sources

bioRxiv2024

KIF2C-induced nuclear condensation concentrates PLK1 and phosphorylated BRCA2 at the kinetochore microtubules in mitosis

During mitosis, the human microtubule depolymerase KIF2C increases the turnover of kinetochore-microtubule attachments. This facilitates the correction of attachment errors. Moreover, BRCA2 phosphorylated at Thr207 by PLK1 (BRCA2-pT207) assembles a complex including PLK1, PP2A and BUBR1 that contributes to the stability of the kinetochore-microtubule attachments. PLK1, together with Aurora B, critically regulate the accurate segregation of chromosomes. Here we demonstrate that KIF2C contains an N-terminal domain that binds directly to several phosphorylated peptides, including BRCA2-pT207. Using an optogenetic platform, we reveal that KIF2C assembles into membrane-less compartments or biomolecular condensates that are located next to microtubules. We provide evidence that condensate assembly depends on the presence of the newly defined N-terminal phospho-binding domain of KIF2C and on the kinase activities of Aurora B and PLK1. Moreover, KIF2C condensates concentrate active PLK1 and colocalize with BRCA2-pT207. We propose that, because of its phospho-dependent binding and oligomerization capacities, KIF2C forms biomolecular condensates that partition PLK1 and locally amplify its kinase activity during mitosis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/589357v1_ufig1.gif" ALT="Figure 1"> View larger version (80K): org.highwire.dtl.DTLVardef@3ff5bforg.highwire.dtl.DTLVardef@11efbdcorg.highwire.dtl.DTLVardef@1295118org.highwire.dtl.DTLVardef@18b3dd3_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

53BP1 interacts with the RNA primer from Okazaki fragments to support their processing during unperturbed DNA replication

RNA-binding proteins are found at replication forks, but their direct interaction with DNA-embedded RNA species that inevitably shape physiological DNA replication remains unexplored. Here we report that 53BP1, involved in the DNA damage and replication stress response, is an RNA-binding protein that directly interacts with Okazaki fragments, in the absence of any external stress. The bulk chromatin association of 53BP1 shows dramatic dependence on PRIM1, which synthesizes the RNA primer of Okazaki fragments. The direct recruitment of 53BP1 to nascent DNA shows susceptibility to in situ ribonuclease A treatment. Conversely, depletion of FEN1, which results in the accumulation of uncleaved RNA primers, leads to an upregulation of 53BP1 levels at the replication forks, suggesting that RNA primers contribute to the recruitment of 53BP1 at the lagging DNA strand. 53BP1 depletion induces an accumulation of S phase poly(ADP-ribose), which constitutes a sensor of unligated Okazaki fragments. Collectively, our data indicate that 53BP1, distinct from its canonical mode of chromatin-binding, is anchored at the replication fork through its RNA-binding activity, highlighting the role of an RNA-protein interaction at DNA replication forks.

molecular biology↗

bioRxiv2022

Identification of key residues of the DNA glycosylase OGG1 controlling efficient DNA scanning and recruitment to oxidized bases in living cells

The DNA-glycosylase OGG1 oversees the detection and clearance of the 7,8-dihydro-8-oxoguanine (8-oxoG), which is the most frequent form of oxidized base in the genome. This lesion is deeply buried within the double-helix and its detection requires careful inspection of the bases by OGG1 via a mechanism that remains only partially understood. By analyzing OGG1 dynamics in the nucleus of living human cells, we demonstrate that the glycosylase constantly scans the DNA by rapidly alternating between diffusion within the nucleoplasm and short transits on the DNA. This scanning process, that we find to be tightly regulated by the conserved residue G245, is crucial for the rapid recruitment of OGG1 at oxidative lesions induced by laser micro-irradiation. Furthermore, we show that residues Y203, N149 and N150, while being all involved in early stages of 8-oxoG probing by OGG1 based on previous structural data, differentially regulate the scanning of the DNA and recruitment to oxidative lesions.

cell biology↗

bioRxiv2021

Sir3 Heterochromatin Protein Promotes NHEJ by Direct Inhibition of Sae2

Heterochromatin is a conserved feature of eukaryotic chromosomes, with central roles in gene expression regulation and maintenance of genome stability. How heterochromatin proteins regulate DNA repair remains poorly described. In Saccharomyces cerevisiae, the Silent Information Regulator (SIR) complex assembles heterochromatin-like chromatin at subtelomeres. SIR-mediated repressive chromatin limits double strand break (DSB) resection protecting damaged chromosome ends during HR. As resection initiation marks the cross-road between repair by non-homologous end joining (NHEJ) or HR, we asked whether SIR- mediated heterochromatin regulates NHEJ. We show that SIRs promote NHEJ through two pathways, one depending on repressive chromatin assembly, and the other relying on Sir3 in a manner that is independent of its heterochromatin-promoting function. Sir3 physically interacts with Sae2 and this interaction impairs Sae2-dependent MRX functions. As a consequence, Sir3 limits Mre11-mediated resection, delays MRX removal from DSB ends and promotes NHEJ.

genetics↗