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Characterization of proliferating cell nuclear antigen (PCNA) from pathogenic yeast Candida albicans and its functional analyses in S. Cerevisiae.

Manohar K, Acharya N - BMC Microbiol. (2015)

Bottom Line: Plasmid segregation in genomic knock out yeast strains showed that CaPCNA but not its G178S mutant complemented for cell survival.Interestingly, wild type strains of C. albicans showed remarkable tolerance to DNA damaging agents when compared with similarly treated yeast cells.Despite structural and physiochemical similarities; we have demonstrated that there are distinct functional differences between ScPCNA and CaPCNA, and probably the ways both the strains maintain their genomic stability.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genomic Instability and Diseases, Department of Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar, 751023, India.

ABSTRACT

Background: Proliferating cell nuclear antigen (PCNA/POL30) an essential protein forms a homotrimeric ring encircling dsDNA and serves as a molecular scaffold to recruit various factors during DNA replication, repair and recombination. According to Candida Genome Database (CGD), orf19.4616 sequence is predicted to encode C. albicans PCNA (CaPCNA) that has not been characterized yet.

Results: Molecular modeling studies of orf19.4616 using S. cerevisiae PCNA sequence (ScPCNA) as a template, and its subsequent biochemical characterizations suggest that like other eukaryotic PCNAs, orf19.4616 encodes for a conventional homotrimeric sliding clamp. Further we showed by surface plasmon resonance that CaPCNA physically interacted with yeast DNA polymerase eta. Plasmid segregation in genomic knock out yeast strains showed that CaPCNA but not its G178S mutant complemented for cell survival. Unexpectedly, heterologous expression of CaPCNA in S. cerevisiae exhibited slow growth phenotypes, sensitivity to cold and elevated temperatures; and showed enhanced sensitivity to hydroxyurea and various DNA damaging agents in comparison to strain bearing ScPCNA. Interestingly, wild type strains of C. albicans showed remarkable tolerance to DNA damaging agents when compared with similarly treated yeast cells.

Conclusions: Despite structural and physiochemical similarities; we have demonstrated that there are distinct functional differences between ScPCNA and CaPCNA, and probably the ways both the strains maintain their genomic stability. We propose that the growth of pathogenic C. albicans which is evolved to tolerate DNA damages could be controlled effectively by targeting this unique fungal PCNA.

No MeSH data available.


Related in: MedlinePlus

Complementation analysis of PCNAs: a. YTS9 strains (trp+) containing various plasmids were selected on synthetic media without tryptophan and uracil. These transformants were grown only on liquid media omitting uracil at 30 °C to cure the resident plasmid as described in methods. After seven such consecutive sub-culturing, presence of the nutritional markers were tested by plating on SD agar plates lacing uracil alone, or tryptophan alone. Plates were incubated for 2 days at 30 °C and photographed. Sectors 1 & 7, empty vectors; 2, 2 μ, ADH1p-CaPCNA, URA3; 3, 2 μ, ADH1p-CaPCNA G178S, URA3; 4, 2 μ, ADH1p-ScPCNA, URA3; 5, 2 μ, CaPCNA gene, URA3; 6, 2 μ, CaPCNA gene G178S, URA3; 8, CEN, CaPCNAgene, URA3; and 9, CEN, CaPCNAgene G178S, URA3. b. The transformants of yeast strain YNA05 (ura+) containing various plasmids were selected on SD media w/o leucine and uracil. Isolated colonies were picked and re-streaked on SD-leucine plate but with or without 5-FOA. Sectors 1, empty vector; 2, 2 μ, ADH1p-ScPCNA G178S, LEU2; 3, 2 μ, ADH1p-ScPCNA, LEU2; 4, 2 μ, ADH1p-CaPCNA, LEU2; 5, 2 μ, CaPCNA G178S, LEU2; 6, 2 μ,CaPCNA gene, LEU2.
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Fig6: Complementation analysis of PCNAs: a. YTS9 strains (trp+) containing various plasmids were selected on synthetic media without tryptophan and uracil. These transformants were grown only on liquid media omitting uracil at 30 °C to cure the resident plasmid as described in methods. After seven such consecutive sub-culturing, presence of the nutritional markers were tested by plating on SD agar plates lacing uracil alone, or tryptophan alone. Plates were incubated for 2 days at 30 °C and photographed. Sectors 1 & 7, empty vectors; 2, 2 μ, ADH1p-CaPCNA, URA3; 3, 2 μ, ADH1p-CaPCNA G178S, URA3; 4, 2 μ, ADH1p-ScPCNA, URA3; 5, 2 μ, CaPCNA gene, URA3; 6, 2 μ, CaPCNA gene G178S, URA3; 8, CEN, CaPCNAgene, URA3; and 9, CEN, CaPCNAgene G178S, URA3. b. The transformants of yeast strain YNA05 (ura+) containing various plasmids were selected on SD media w/o leucine and uracil. Isolated colonies were picked and re-streaked on SD-leucine plate but with or without 5-FOA. Sectors 1, empty vector; 2, 2 μ, ADH1p-ScPCNA G178S, LEU2; 3, 2 μ, ADH1p-ScPCNA, LEU2; 4, 2 μ, ADH1p-CaPCNA, LEU2; 5, 2 μ, CaPCNA G178S, LEU2; 6, 2 μ,CaPCNA gene, LEU2.

Mentions: POL30 gene in any organism is indispensable for cell survival. Because our biochemical and structural modelling studies suggested CaPCNA to be alike of ScPCNA, it compelled us to examine whether CaPCNA can perform the essential functions of yeast POL30 and support cell viability of the genomic strain. To achieve our goal CaPCNA was expressed either under its own promoter or under a yeast constitutive promoter ADH1 in genomic deletion strains of S. cerevisiae but survives due to presence of a plasmid bearing ScPCNA. The segregation of plasmids was carried out to replace the ScPCNA with CaPCNA by two different approaches. In one approach, YTS9 strain which is for genomic POL30 but carrying YCP-ScPOL30 DE41, 42AA-TRP1 plasmid for survival was used to obtain transformants harbouring various CaPCNA plasmids (URA3) on SD media lacking tryptophan and uracil. Attempt to cure the retained plasmid was carried out by repeated sub-culturing of the transformants on SD-uracil liquid media as described in methods. About 30 isolated colonies from each set were streaked on SD-uracil and SD-tryptophan plates. The percentage of curing of resident plasmid was scored and a representative figure has been shown (Fig. 6 A). The lack of growth on SD-tryptophan plate but growth on SD-uracil will suggest complete curing of the resident PCNA and complementation due to incoming plasmid. Strains containing CaPCNA or its gene in both CEN/2 μ vectors were able to replace the resident plasmid (Fig. 6 A sectors 2, 5 and 8), and about 100 % efficient curing was achievable. Similar result was also obtained for incoming ScPCNA but not for the vector controls as both the nutritional marker phenotypes (URA and TRP) were retained (Fig. 6 A compare sector 4 with 1 and 7).Fig. 6


Characterization of proliferating cell nuclear antigen (PCNA) from pathogenic yeast Candida albicans and its functional analyses in S. Cerevisiae.

Manohar K, Acharya N - BMC Microbiol. (2015)

Complementation analysis of PCNAs: a. YTS9 strains (trp+) containing various plasmids were selected on synthetic media without tryptophan and uracil. These transformants were grown only on liquid media omitting uracil at 30 °C to cure the resident plasmid as described in methods. After seven such consecutive sub-culturing, presence of the nutritional markers were tested by plating on SD agar plates lacing uracil alone, or tryptophan alone. Plates were incubated for 2 days at 30 °C and photographed. Sectors 1 & 7, empty vectors; 2, 2 μ, ADH1p-CaPCNA, URA3; 3, 2 μ, ADH1p-CaPCNA G178S, URA3; 4, 2 μ, ADH1p-ScPCNA, URA3; 5, 2 μ, CaPCNA gene, URA3; 6, 2 μ, CaPCNA gene G178S, URA3; 8, CEN, CaPCNAgene, URA3; and 9, CEN, CaPCNAgene G178S, URA3. b. The transformants of yeast strain YNA05 (ura+) containing various plasmids were selected on SD media w/o leucine and uracil. Isolated colonies were picked and re-streaked on SD-leucine plate but with or without 5-FOA. Sectors 1, empty vector; 2, 2 μ, ADH1p-ScPCNA G178S, LEU2; 3, 2 μ, ADH1p-ScPCNA, LEU2; 4, 2 μ, ADH1p-CaPCNA, LEU2; 5, 2 μ, CaPCNA G178S, LEU2; 6, 2 μ,CaPCNA gene, LEU2.
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Related In: Results  -  Collection

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Fig6: Complementation analysis of PCNAs: a. YTS9 strains (trp+) containing various plasmids were selected on synthetic media without tryptophan and uracil. These transformants were grown only on liquid media omitting uracil at 30 °C to cure the resident plasmid as described in methods. After seven such consecutive sub-culturing, presence of the nutritional markers were tested by plating on SD agar plates lacing uracil alone, or tryptophan alone. Plates were incubated for 2 days at 30 °C and photographed. Sectors 1 & 7, empty vectors; 2, 2 μ, ADH1p-CaPCNA, URA3; 3, 2 μ, ADH1p-CaPCNA G178S, URA3; 4, 2 μ, ADH1p-ScPCNA, URA3; 5, 2 μ, CaPCNA gene, URA3; 6, 2 μ, CaPCNA gene G178S, URA3; 8, CEN, CaPCNAgene, URA3; and 9, CEN, CaPCNAgene G178S, URA3. b. The transformants of yeast strain YNA05 (ura+) containing various plasmids were selected on SD media w/o leucine and uracil. Isolated colonies were picked and re-streaked on SD-leucine plate but with or without 5-FOA. Sectors 1, empty vector; 2, 2 μ, ADH1p-ScPCNA G178S, LEU2; 3, 2 μ, ADH1p-ScPCNA, LEU2; 4, 2 μ, ADH1p-CaPCNA, LEU2; 5, 2 μ, CaPCNA G178S, LEU2; 6, 2 μ,CaPCNA gene, LEU2.
Mentions: POL30 gene in any organism is indispensable for cell survival. Because our biochemical and structural modelling studies suggested CaPCNA to be alike of ScPCNA, it compelled us to examine whether CaPCNA can perform the essential functions of yeast POL30 and support cell viability of the genomic strain. To achieve our goal CaPCNA was expressed either under its own promoter or under a yeast constitutive promoter ADH1 in genomic deletion strains of S. cerevisiae but survives due to presence of a plasmid bearing ScPCNA. The segregation of plasmids was carried out to replace the ScPCNA with CaPCNA by two different approaches. In one approach, YTS9 strain which is for genomic POL30 but carrying YCP-ScPOL30 DE41, 42AA-TRP1 plasmid for survival was used to obtain transformants harbouring various CaPCNA plasmids (URA3) on SD media lacking tryptophan and uracil. Attempt to cure the retained plasmid was carried out by repeated sub-culturing of the transformants on SD-uracil liquid media as described in methods. About 30 isolated colonies from each set were streaked on SD-uracil and SD-tryptophan plates. The percentage of curing of resident plasmid was scored and a representative figure has been shown (Fig. 6 A). The lack of growth on SD-tryptophan plate but growth on SD-uracil will suggest complete curing of the resident PCNA and complementation due to incoming plasmid. Strains containing CaPCNA or its gene in both CEN/2 μ vectors were able to replace the resident plasmid (Fig. 6 A sectors 2, 5 and 8), and about 100 % efficient curing was achievable. Similar result was also obtained for incoming ScPCNA but not for the vector controls as both the nutritional marker phenotypes (URA and TRP) were retained (Fig. 6 A compare sector 4 with 1 and 7).Fig. 6

Bottom Line: Plasmid segregation in genomic knock out yeast strains showed that CaPCNA but not its G178S mutant complemented for cell survival.Interestingly, wild type strains of C. albicans showed remarkable tolerance to DNA damaging agents when compared with similarly treated yeast cells.Despite structural and physiochemical similarities; we have demonstrated that there are distinct functional differences between ScPCNA and CaPCNA, and probably the ways both the strains maintain their genomic stability.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genomic Instability and Diseases, Department of Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar, 751023, India.

ABSTRACT

Background: Proliferating cell nuclear antigen (PCNA/POL30) an essential protein forms a homotrimeric ring encircling dsDNA and serves as a molecular scaffold to recruit various factors during DNA replication, repair and recombination. According to Candida Genome Database (CGD), orf19.4616 sequence is predicted to encode C. albicans PCNA (CaPCNA) that has not been characterized yet.

Results: Molecular modeling studies of orf19.4616 using S. cerevisiae PCNA sequence (ScPCNA) as a template, and its subsequent biochemical characterizations suggest that like other eukaryotic PCNAs, orf19.4616 encodes for a conventional homotrimeric sliding clamp. Further we showed by surface plasmon resonance that CaPCNA physically interacted with yeast DNA polymerase eta. Plasmid segregation in genomic knock out yeast strains showed that CaPCNA but not its G178S mutant complemented for cell survival. Unexpectedly, heterologous expression of CaPCNA in S. cerevisiae exhibited slow growth phenotypes, sensitivity to cold and elevated temperatures; and showed enhanced sensitivity to hydroxyurea and various DNA damaging agents in comparison to strain bearing ScPCNA. Interestingly, wild type strains of C. albicans showed remarkable tolerance to DNA damaging agents when compared with similarly treated yeast cells.

Conclusions: Despite structural and physiochemical similarities; we have demonstrated that there are distinct functional differences between ScPCNA and CaPCNA, and probably the ways both the strains maintain their genomic stability. We propose that the growth of pathogenic C. albicans which is evolved to tolerate DNA damages could be controlled effectively by targeting this unique fungal PCNA.

No MeSH data available.


Related in: MedlinePlus