TheRAD52ortholog ofYarrowia lipolyticais essential for nuclear integrity and DNA repair

Abstract

Yarrowia lipolytica (Yl) is a dimorphic fungus that has become a well-established model for a number of biological processes, including secretion of heterologous and chimerical proteins. However, little is known on the recombination machinery responsible for the integration in the genome of the exogenous DNA encoding for those proteins. We have carried out a phenotypic analysis of rad52 deletants of Y. lipolytica. YlRad52 exhibited 20-30% identity with Rad52 homologues of other eukaryotes, including Saccharomyces cerevisiae and Candida albicans. Ylrad52-Δ strains formed colonies on YPD-agar plates which were spinier and smaller than those from wild type, whereas in YPD liquid cultures they exhibited a decreased grow rate and contained cells with aberrant morphology and fragmented chromatin, supporting a role for homologous recombination (HR) in genome stability under nondamaging conditions. In addition, Ylrad52 mutants showed moderate to high sensitivity to UV light, oxidizing agents and compounds that cause single- (SSB) and double-strand breaks (DSB), indicating an important role for Rad52 in DNA repair. These findings extend to Yl previous observations indicating that RAD52 is a crucial gene for DNA repair in other fungi, including S. cerevisiae, C. albicans and Schizosaccharomyces pombe.

Improving the homologous recombination efficiency of Yarrowia lipolytica by grafting the heterogenous component from Saccharomyces cerevisiae

“…Compared with Po1f-Δku70-ScRad52, the HR efficiency of Po1f-Δku70-ScRad52-ΔYlRad52 was reduced by 92.9%, and there was practically no recombination with homology arms of 250 and 100 bp. These results show that the YlRad52 gene plays a crucial role in the native HR mechanism of Y. lipolytica , which is consistent with the previous report that the Rad52 gene in is crucial for DNA repair in this yeast [26].…”

“…This may be explained by the fact that Rad52 is important for nuclear integrity in Y. lipolytica . This result is similar to a report by Campos-Gongora et al [26]. The expression of the ScRad52 gene alleviates the damage to nuclear integrity caused by the deletion of the endogenous Y. lipolyticaRad52 to some extent.…”

“…Consequently, strengthening the HR repair is also an effective approach for improving the gene targeting efficiency. The HR repair pathway is mediated by a class of conserved enzymes called DNA recombinase enzymes, most of which were initially identified in Saccharomyces cerevisiae [26]. Notably, the RAD51/RAD52 complex is a major participant in the targeted integration of foreign DNA in eukaryotes and plays a key role in the HR pathway [27] (Fig.…”

Metabolic Engineering of Yarrowia lipolytica for Terpenoid Production: Tools and Strategies

“…Direct enhancement of HR is another promising approach to improving the HR efficiency in Y. lipolytica . The key proteins of HR include the RAD52 superfamily (RAD51, RAD52, RAD54, RAD55/57, RDH54, RAD59), exonucleases involved in DSB end cleavage (MRE11-RD50-XRS2 complex, EXO1, SGS1-DNA2), and SAE2 . Among them, the RAD51/RAD52 complex plays a key role in the targeted integration of eukaryotic exogenous DNA .…”

Synthetic biology tools for engineering Yarrowia lipolytica

“…The nucleus can be targeted by fusing the well-known viral SV40 nuclear localization sequence (NLS) (PKKKRKV) to the protein (e.g., for efficiently targeting Cas9; Schwartz et al, 2016 ). Other putative nuclear localization signals ( Campos-Góngora et al, 2013 ) as well as putative mitochondrial targeting sequences ( Bakkaiova et al, 2014 ; Kerscher et al, 2004 ) have been identified for Y. lipolytica proteins. However, no further analyses were done, to our knowledge, to characterize these endogenous targeting sequences.…”