Biology subjects

Madru, C.

Publications and source records attributed to Madru, C..

4 recordsLinked to original sources

bioRxiv2026

Permuted 23S rRNA is integrated in 50S ribosome particles in Thermococcus barophilus

In Archaea, a prevalent class of circular RNAs corresponds to 16S and 23S ribosomal RNA intermediates (circ-pre-rRNAs). A conserved bulge-helix-bulge (BHB) motif within the 16S and 23S rRNAs processing stems and adjacent to the circularization site in Euryarchaeota and TACK superphylum suggests that pre-rRNAs circularization is widely conserved across Archaea. Using genome-wide transcriptomic data obtained on total RNAs of wild-type Thermococcus barophilus cells, we recovered the canonical circularization junctions of the 16S and 23S circ-pre-rRNAs at the predicted BHB motifs. We also identified three alternatives 23S circular junctions introducing variability at the 3 end of the mature rRNA. We investigated the different forms of 16S and 23S by performing primer extension and RACE experiments. We showed that while the 16S rRNA has standard 5 and 3 extremities, the main form of 23S rRNA is circularly permuted, with helix H99 now at its 5 end. This permutation most probably emerged from the deletion of helix H98 from the 23S circ-pre-rRNA. Interestingly, we showed that the permuted 23S rRNA is incorporated into ribosome subunits and 70S monosomes. The significance of this event in generating functional 50S particles remains to be determined.

microbiology↗

bioRxiv2024

Communication between DNA polymerases and Replication Protein A within the archaeal replisome

Replication Protein A (RPA) plays a pivotal role in DNA replication by coating and protecting exposed single-stranded DNA, and acting as a molecular hub that recruits additional replication factors. We demonstrated that archaeal RPA hosts a winged-helix domain (WH) that interacts with two key actors of the replisome: the DNA primase (PriSL) and the replicative DNA polymerase (PolD). Using an integrative structural biology approach, combining nuclear magnetic resonance, X-ray crystallography and cryo-electron microscopy, we unveiled how RPA interacts with PriSL and PolD through two distinct surfaces of the WH domain: an evolutionarily conserved interface and a novel binding site. Finally, RPA was shown to stimulate the activity of PriSL in a WH-dependent manner. This study provides a molecular understanding of the WH-mediated regulatory activity in conserved central replication factors such as RPA, which regulate genome maintenance in Archaea and Eukaryotes.

molecular biology↗

bioRxiv2022

DNA-binding mechanism and evolution of Replication Protein A

Replication Protein A (RPA) is a heterotrimeric single stranded DNA-binding protein with essential roles in DNA replication, recombination and repair, in both eukaryotic and archaeal cells. By using an integrative approach that combines three crystal structures, four cryo-EM structures in complex with single-stranded DNA (ssDNA) of different lengths, we extensively characterized RPA from Pyrococcus abyssi in different states. These structures show two essential features conserved in eukaryotes: a trimeric core and a module that promotes cooperative binding to ssDNA, as well as a newly identified archaeal-specific domain. These structures reveal for the first time how ssDNA is handed over from one RPA complex to the other, and uncover an unanticipated mechanism of self-association on ssDNA tracts. This work constitutes a significant step forward in the molecular understanding of the structure and DNA-binding mechanism of RPA, with far-reaching implications for the evolution of this primordial replication factor in Archaea and Eukarya.

biochemistry↗

bioRxiv2020

Structural basis for the increased processivity of D-family DNA polymerases in complex with PCNA

Replicative DNA polymerases (DNAPs) have evolved the ability to copy the genome with high processivity and fidelity. In Eukarya and Archaea, the processivity of replicative DNAPs is greatly enhanced by its binding to the proliferative cell nuclear antigen (PCNA) that encircles the DNA. We determined the cryo-EM structure of the DNA-bound PolD-PCNA complex from Pyrococcus abyssi at 3.77[A]. Using an integrative structural biology approach - combining cryo-EM, X-ray crystallography and protein-protein interaction measurements - we describe the molecular basis for the interaction and cooperativity between a replicative DNAP and PCNA with an unprecedented level of detail. PolD recruits PCNA via a complex mechanism, which requires two different PIP-boxes. We infer that the second PIP-box, which is shared with the eukaryotic Pol replicative DNAP, plays a dual role in binding either PCNA or primase, and could be a master switch between an initiation phase and a processive phase during replication.

biochemistry↗