Deletion of CnLIG4 DNA ligase gene in the fungal pathogen Cryptococcus neoformans elevates homologous recombination efficiency
Shimizu, K., Chiba Univ. (Japan) | Li, H.M. | Virtudazo, E.V. | Watanabe, A. | Kamei, K. | Yamaguchi, M. | Kawamoto, S.
In eukaryotes from yeasts to human, DNA double-strand breaks are repaired by nonhomologous end-joining (NHEJ) or homologous integration (HI). In the human pathogenic yeast Cryptococcus neoformans, gene manipulation by HI does not occur frequently because ectopic integration by NHEJ is predominant, and it has been necessary to screen 30 - 100 transformants per experiment to obtain transformants with the desired genotypes. To overcome this problem, we constructed a strain in which one of the NHEJ-related genes, CnLIG4, was deleted. CnLIG4 encodes a homologue of the human DNA ligase IV involved in the last step of DNA repair by NHEJ. Gene targeting in the URA5 locus of a URA5-lacking strain TAD1 with URA5 gene fragments having 1-kb flanking sequences achieved 80% HI efficiency, which is higher than that of the wild-type control (50%). Growth phenotypes and virulence were not attenuated by deletion of the CnLIG4 gene. Such results suggest that the CnLIG4 knockout strain created in this study provides an additional alternative for the molecular genetics study of C. neoformans.
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