Noncanonical Roles of RAD51.

Homologous recombination (HR), an evolutionary conserved pathway, plays a paramount role(s) in genome plasticity. The pivotal HR step is the strand invasion/exchange of double-stranded DNA by a homologous single-stranded DNA (ssDNA) covered by RAD51. Thus, RAD51 plays a prime role in HR through this canonical catalytic strand invasion/exchange activity. The mutations in many HR genes cause oncogenesis. Surprisingly, despite its central role in HR, the invalidation of RAD51 is not classified as being cancer prone, constituting the "RAD51 paradox". This suggests that RAD51 exercises other noncanonical roles that are independent of its catalytic strand invasion/exchange function. For example, the binding of RAD51 on ssDNA prevents nonconservative mutagenic DNA repair, which is independent of its strand exchange activity but relies on its ssDNA occupancy. At the arrested replication forks, RAD51 plays several noncanonical roles in the formation, protection, and management of fork reversal, allowing for the resumption of replication. RAD51 also exhibits noncanonical roles in RNA-mediated processes. Finally, RAD51 pathogenic variants have been described in the congenital mirror movement syndrome, revealing an unexpected role in brain development. In this review, we present and discuss the different noncanonical roles of RAD51, whose presence does not automatically result in an HR event, revealing the multiple faces of this prominent actor in genomic plasticity.

A proteomic screen of Ty1 integrase partners identifies the protein kinase CK2 as a regulator of Ty1 retrotransposition.

BACKGROUND: Transposable elements are ubiquitous and play a fundamental role in shaping genomes during evolution. Since excessive transposition can be mutagenic, mechanisms exist in the cells to keep these mobile elements under control. Although many cellular factors regulating the mobility of the retrovirus-like transposon Ty1 in Saccharomyces cerevisiae have been identified in genetic screens, only very few of them interact physically with Ty1 integrase (IN). RESULTS: Here, we perform a proteomic screen to establish Ty1 IN interactome. Among the 265 potential interacting partners, we focus our study on the conserved CK2 kinase. We confirm the interaction between IN and CK2, demonstrate that IN is a substrate of CK2 in vitro and identify the modified residues. We find that Ty1 IN is phosphorylated in vivo and that these modifications are dependent in part on CK2. No significant change in Ty1 retromobility could be observed when we introduce phospho-ablative mutations that prevent IN phosphorylation by CK2 in vitro. However, the absence of CK2 holoenzyme results in a strong stimulation of Ty1 retrotransposition, characterized by an increase in Ty1 mRNA and protein levels and a high accumulation of cDNA. CONCLUSION: Our study shows that Ty1 IN is phosphorylated, as observed for retroviral INs and highlights an important role of CK2 in the regulation of Ty1 retrotransposition. In addition, the proteomic approach enabled the identification of many new Ty1 IN interacting partners, whose potential role in the control of Ty1 mobility will be interesting to study.

Ty1 integrase is composed of an active N-terminal domain and a large disordered C-terminal module dispensable for its activity in vitro.

Long-terminal repeat (LTR) retrotransposons are genetic elements that, like retroviruses, replicate by reverse transcription of an RNA intermediate into a complementary DNA (cDNA) that is next integrated into the host genome by their own integrase. The Ty1 LTR retrotransposon has proven to be a reliable working model to investigate retroelement integration site preference. However, the low yield of recombinant Ty1 integrase production reported so far has been a major obstacle for structural studies. Here we analyze the biophysical and biochemical properties of a stable and functional recombinant Ty1 integrase highly expressed in E.coli. The recombinant protein is monomeric and has an elongated shape harboring the three-domain structure common to all retroviral integrases at the N-terminal half, an extra folded region, and a large intrinsically disordered region at the C-terminal half. Recombinant Ty1 integrase efficiently catalyzes concerted integration in vitro, and the N-terminal domain displays similar activity. These studies that will facilitate structural analyses may allow elucidating the molecular mechanisms governing Ty1 specific integration into safe places in the genome.