Biology subjects

Zamarreno, J.

Publications and source records attributed to Zamarreno, J..

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↗

bioRxiv2022

The AAA+ ATPase RavA-ViaA complex sensitizes Escherichia coli to aminoglycosides under anaerobic low energy conservation conditions

Aminoglycosides have been used against Gram-negative bacteria for decades. Yet, uncertainties remain about various aspects of their uptake mechanism. Moreover their killing efficiency is well known to vary as a function of growth conditions and types of metabolism used by the targeted bacterium. Here we show that RavA, an AAA+ ATPase from the MoxR subfamily, associated with its VWA-containing partner, ViaA sensitize E. coli to lethal concentrations of AG, including gentamycin (Gm) and tobramycin, but not of antibiotics of other classes. We show this sensitizing effect to be due to enhanced Gm uptake in a proton motive force dependent manner. We evaluated the influence of RavA ViaA throughout a series of growth conditions, including aerobiosis and anaerobiosis. This led us to observe that the sensitizing effect of RavA ViaA varies with the respiratory chain used, i.e. RavA ViaA influence was prominent in the absence of exogenous electron acceptor or with fumarate, i.e. in poor energy conservation conditions, and dispensable in the presence of nitrate or oxygen, i.e. in high level of energy conservation. We propose RavA ViaA to be able to sense energetic state of the cell and to be used under low energy conditions for facilitating uptake of chemicals across the membrane, including Gm. Author SummaryAntibiotic resistance is a major public health, social and economic problem. Aminoglycosides are known for their high efficiency against Gram-negative bacteria but their use is restricted to life threatening infections because of their nephrotoxicity and ototoxicity at therapeutic dose. Elucidation of AG sensitization mechanisms in bacteria will allow the use of a decreased effective dose of AGs. Here we identified new molecular actors, RavA and ViaA, which sensitize E. coli to AG under anaerobiosis. RavA belongs to the AAA+ ATPase family while ViaA bears a VWA motif. Moreover we show here that the influence of RavA ViaA on AG sensitivity varies with growth conditions and respiratory metabolism used by E. coli. This is a significant step forward as anaerobiosis is well known to reduce antibacterial activity of AG. This study emphasizes the crucial importance of the relationships between culture conditions, metabolism and antibiotic resistance.

microbiology↗