Biology subjects

Bueno, A.

Publications and source records attributed to Bueno, A..

4 recordsLinked to original sources

bioRxiv2024

Strand asymmetry of DNA damage tolerance mechanisms

DNA damage tolerance mechanisms are crucial for timely and accurate chromosomal replication in response to DNA polymerase stalling. Ubiquitylation of the replicative sliding clamp PCNA drives major tolerance pathways, error-free homologous recombination template switching and error-prone translesion synthesis, though their dynamics at forks and pathway choice determinants are poorly understood. Using strand-specific genomics we revealed an asymmetric nature of tolerance pathways, characterized by preferential template switching-driven recombinase engagement of stalled nascent lagging strands and translesion synthesis usage in response to leading strand polymerase stalling. This asymmetry, determined by a strand-dynamic interplay between PCNA-ubiquitin writers and erasers, likely stems from necessities dictated by leading and lagging strand replication mechanisms and has implications for asymmetric mutation inheritance. One-Sentence SummaryDNA damage tolerance mechanisms respond asymmetrically to leading or lagging strand polymerase blocks.

molecular biology↗

bioRxiv2024

Timely lagging strand maturation relies on Ubp10-mediated PCNA dissociation from replicating chromatin

Synthesis and maturation of Okazaki Fragments is an incessant and highly efficient metabolic process completing the synthesis of the lagging strands at replication forks during S phase. Accurate Okazaki fragment maturation (OFM) is crucial to maintain genome integrity and, therefore, cell survival in all living organisms. In eukaryotes, OFM involves the consecutive action of DNA polymerase Pol {partial}, 5 Flap endonuclease Fen1 and DNA ligase I, and constitutes the best example of a sequential process coordinated by the sliding clamp PCNA. For OFM to occur efficiently, cooperation of these enzymes with PCNA must be highly regulated. Here, we present evidence of a role for the PCNA-deubiquitylase Ubp10 in the maturation of Okazaki fragments in the budding yeast Saccharomyces cerevisiae. We show that Ubp10 associates with lagging-strand DNA synthesis machineries on replicating chromatin to ensure timely ligation of Okazaki fragments by promoting an Elg1ATAD5-independent PCNA unloading mechanism. This document was written without the use of AI.

molecular biology↗

bioRxiv2023

Ubiquitin protease Ubp1 cooperates with Ubp10 and Ubp12 to revert Lysine-164 PCNA ubiquitylation at replication forks

Proliferating cell nuclear antigen (PCNA) is essential for the faithful duplication of eukaryotic genomes. PCNA orchestrates events necessary to deal with threats to genomic integrity, such as the DNA damage tolerance (DDT) response. DDT is a mechanism by which eukaryotic cells bypass replication-blocking lesions to prevent replisome instability. DDT pathways are regulated by the ubiquitylation of PCNA and the consequent recruitment of specialized polymerases and mechanisms able to guarantee the continuity of replication. We have previously described that the deubiquitylases Ubp10 and Ubp12 associate with replication forks and modulate DDT events by reverting the ubiquitylation of PCNA in Saccharomyces cerevisiae. The results of this study unveil Ubp1 as a new PCNA deubiquitylase, which cooperates with Ubp10 and Ubp12 in the regulation of DDT during DNA replication. Ubp1 is known as a cytoplasmic protein, however, we found that it also localizes to the nucleus where it binds to chromatin and associates with DNA replication forks. In addition, Ubp1 interacts with and deubiquitylates PCNA. The ablation of Ubp1, Ubp10, and Ubp12 enhances both the accumulation of ubiquitylated PCNA and the DNA replication defects observed in cells depleted for Ubp10 and Ubp12, supporting a cooperative role among the three enzymes. IMPORTANCEPCNA ubiquitylation regulates DDT mechanisms to bypass genotoxic lesions during replication and that PCNA deubiquitylation is required to limit the extent of bypass events. This study shows that Saccharomyces cerevisiae PCNA is ubiquitylated during an unperturbed S-phase progression and that three ubiquitin proteases (Ubp1, Ubp10, and Ubp12) work together facilitating DNA replication by efficiently controlling ubiquitylation of PCNA at replication forks.

molecular biology↗

bioRxiv2018

Biochemical analyses reveal amino acid residues critical for cell cycle-dependent phosphorylation of human Cdc14A phosphatase by cyclin-dependent kinase 1

Cdc14 enzymes compose a family of highly conserved phosphatases that are present in a wide range of organisms, including yeast and humans, and that preferentially reverse the phosphorylation of Cyclin-Dependent Kinase (Cdk) substrates. The budding yeast Cdc14 orthologue has essential functions in the control of late mitosis and cytokinesis. In mammals, however, the two Cdc14 homologues, Cdc14A and Cdc14B, do not play a prominent role in controlling late mitotic events, suggesting that some Cdc14 functions are not conserved across species. Moreover, in yeast, Cdc14 is regulated by changes in its subcellular location and by phosphorylation events. In contrast, little is known about the regulation of human Cdc14 phosphatases. Here, we have studied how the human Cdc14A orthologue is regulated during the cell cycle. We found that Cdc14A is phosphorylated on Ser411, Ser453 and Ser549 by Cdk1 early in mitosis and becomes dephosphorylated during late mitotic stages. Interestingly, in vivo and in vitro experiments revealed that, unlike in yeast, Cdk1-mediated phosphorylation of human Cdc14A did not control its catalytic activity but likely modulated its interaction with other proteins in early mitosis. These findings point to differences in Cdk1-mediated mechanisms of regulation between human and yeast Cdc14 orthologues.

biochemistry↗