The G protein ␣ subunits Gpa1, Gpa2, and Gpa3 mediate signal transduction and are important in the growth and virulence of Cryptococcus neoformans. To understand how Gpa1 functions without a conventional G subunit, we characterized a resistance to inhibitors of cholinesterase 8 (Ric8) homolog from C. neoformans, which shares amino acid sequence homology with other Ric8 proteins that exhibit guanine nucleotide exchange factor (GEF) activity toward G␣. We found that the ric8 mutant was reduced in capsule size and melanin formation, which could be suppressed by cyclic AMP (cAMP) supplementation or by introducing the activated GPA1 Q284L allele. Consistent with the fact that Ric8 participates in cAMP signaling to regulate virulence, the ric8 mutant was attenuated in virulence toward mice. Interestingly, disruption of RIC8 also resulted in opposing effects on pheromone signaling, as the ric8 mutant showed reduced mating but an enhanced ability to induce the pheromone response in the mating partner. To identify Ric8 functional mechanisms, we examined the interactions between Ric8 and the three G␣ proteins. Ric8 interacted with Gpa1 and Gpa2, but not Gpa3. The presence of Gpa1 Q284L negatively affected its interaction with Ric8, whereas the activated Gpa2 Q203L allele abolished the interaction. Collectively, these findings suggest that Ric8 functions as a GEF to facilitate the activation of Gpa1-cAMP signaling and to promote Gpa2, affecting mating efficiency. Our study highlights the distinct and conserved characteristics associated with G protein signaling and contributes to our overall understanding of how G protein ␣ subunits function with or without a canonical G partner in C. neoformans.
The collection of gene deletion mutants of Saccharomyces cerevisiae was used to screen for novel genes required for UV-induced mutagenesis. We found the SBF transcription factor (Swi4/Swi6 protein complex) to be required for wild-type levels of UV mutability in forward and reverse mutation assay. Expression of translesion polymerase ζ component Rev7 was identified as a target of SBFdependent regulation.
KeywordsYeast; UV Radiation; Mutagenesis; Translesion synthesis; Transcriptional regulation Genetic instability resulting from enhanced mutagenesis is a severe consequence of exposing pro-and eukaryotic cells to DNA-damaging agents [1]. Especially for bulky adducts, such as UV-C induced pyrimidine dimers, point mutations opposite DNA lesions of reduced coding capacity mainly arise through active processes that ensure the completion of replication of a damaged template. In recent years, it became clear that high-fidelity replicative DNA polymerases are temporarily replaced at the lesion site by one of several translesion polymerases whose active sites can accommodate modified bases while lacking proofreading activity [2,3]. A concerted action of one polymerase inserting a base opposite a lesion together with another polymerase extending from the imperfectly matched primer/template junction may be required [4]. The ensuing damage bypass may be error-free or error-prone, depending on type of lesion and polymerases involved. Most of the enhanced mutability following UV radiation is generated by such error-prone translesion synthesis. The majority of UV-induced and spontaneous mutations are dependent on polymerase ζ, a complex of .Our present understanding of the events leading to mutagenic translesion synthesis also assigns a major role to proliferating cell nuclear antigen (PCNA). PCNA is subject to ubiquitination by the Rad6/Rad18 complex [8]. Monoubiquitinated PCNA appears to facilitate the switch from replicative to bypass polymerase [9,10] and thus, ubiquitin binding domains of bypass polymerases were found to be important for function [11][12][13][14]. Mutation of PCNA that prevents ubiquitination abolishes most of UV-induced mutability [15,16] Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. The now commercially available collection of deletion mutants of non-essential yeast genes has previously been exploited to screen for mutants with enhanced spontaneous mutability and gross chromosomal rearrangements [28,29]. Here, we used this collection to identify novel genes required for UV-induced mutagenesis that may have been missed previously. The identical approach h...
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