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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

Sensitivities of wild type C. albicans SC5314 and S. cerevisiae EMY74.7 to HU and DNA damaging agents. Cells from an overnight YPD + uridine culture were serially diluted and spotted onto YPD + uridine plates without or with indicated concentrations of HU, MMS, H2O2 and TBHP. The plates were incubated at 30 °C for 3 days and then photographed.
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Fig9: Sensitivities of wild type C. albicans SC5314 and S. cerevisiae EMY74.7 to HU and DNA damaging agents. Cells from an overnight YPD + uridine culture were serially diluted and spotted onto YPD + uridine plates without or with indicated concentrations of HU, MMS, H2O2 and TBHP. The plates were incubated at 30 °C for 3 days and then photographed.

Mentions: Heterologous expression of CaPCNA in non-pathogenic S. cerevisiae resulted in sensitivity to DNA damaging agents, however during infection C. albicans evades various attacks of ROS/RNI species generated by innate immune cells [36]. Whether in a homologous system CaPCNA provides any protective role, a comparative study of susceptibility by wild type strains of C. albicans and yeast to DNA damaging agents was carried out (Fig. 9). When equal number cells were diluted and spotted; interestingly, C. albicans strain SC5314 which is a commonly used laboratory wild type strain overgrew S. cerevisiae strain EMY74.7, a derivative of widely used DBY747. So the retardation of growth rate in Fig. 7A where we checked growth rates of yeast containing CaPCNA is not due to the characteristic CaPCNA rather because of lack of species specific interaction with replication components. Despite the differences in the growth rate, Candida SC5314 exhibited moderate sensitivity to HU than yeast EMY74.7 at 50 mM HU, the reason for such a phenotype requires further extensive analysis (Fig. 9 A). The C. albicans SC5314 showed tolerance to DNA damaging agents like MMS, H2O2, and TBHP unlike yeast strain expressing CaPCNA; and at same or lower concentration of reagents, a significant impairment of growth of EMY74.7 was observed. In a different study, similar results have been reported earlier by using diploid strains of S. cerevisiae BY4743 and C. albicans DKCa39 [29, 30]. While one copy of PCNA gene can be easily deleted from diploid S. cerevisiae, because of haplo-insufficiency similar deletion was not achievable in C. albicans [37, 38]. Taken all together we suggest that the fungal pathogen C. albicans is genetically evolved to be resistant to DNA damages in comparison to nonpathogenic budding yeast; and CaPCNA dependent DNA damage tolerance pathways could play a crucial role in addition to other cellular mechanisms like efficient drug efflux pumps, thicker cell wall and any other components present in C. albicans to maintain genomic stability.Fig. 9


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)

Sensitivities of wild type C. albicans SC5314 and S. cerevisiae EMY74.7 to HU and DNA damaging agents. Cells from an overnight YPD + uridine culture were serially diluted and spotted onto YPD + uridine plates without or with indicated concentrations of HU, MMS, H2O2 and TBHP. The plates were incubated at 30 °C for 3 days and then photographed.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4634812&req=5

Fig9: Sensitivities of wild type C. albicans SC5314 and S. cerevisiae EMY74.7 to HU and DNA damaging agents. Cells from an overnight YPD + uridine culture were serially diluted and spotted onto YPD + uridine plates without or with indicated concentrations of HU, MMS, H2O2 and TBHP. The plates were incubated at 30 °C for 3 days and then photographed.
Mentions: Heterologous expression of CaPCNA in non-pathogenic S. cerevisiae resulted in sensitivity to DNA damaging agents, however during infection C. albicans evades various attacks of ROS/RNI species generated by innate immune cells [36]. Whether in a homologous system CaPCNA provides any protective role, a comparative study of susceptibility by wild type strains of C. albicans and yeast to DNA damaging agents was carried out (Fig. 9). When equal number cells were diluted and spotted; interestingly, C. albicans strain SC5314 which is a commonly used laboratory wild type strain overgrew S. cerevisiae strain EMY74.7, a derivative of widely used DBY747. So the retardation of growth rate in Fig. 7A where we checked growth rates of yeast containing CaPCNA is not due to the characteristic CaPCNA rather because of lack of species specific interaction with replication components. Despite the differences in the growth rate, Candida SC5314 exhibited moderate sensitivity to HU than yeast EMY74.7 at 50 mM HU, the reason for such a phenotype requires further extensive analysis (Fig. 9 A). The C. albicans SC5314 showed tolerance to DNA damaging agents like MMS, H2O2, and TBHP unlike yeast strain expressing CaPCNA; and at same or lower concentration of reagents, a significant impairment of growth of EMY74.7 was observed. In a different study, similar results have been reported earlier by using diploid strains of S. cerevisiae BY4743 and C. albicans DKCa39 [29, 30]. While one copy of PCNA gene can be easily deleted from diploid S. cerevisiae, because of haplo-insufficiency similar deletion was not achievable in C. albicans [37, 38]. Taken all together we suggest that the fungal pathogen C. albicans is genetically evolved to be resistant to DNA damages in comparison to nonpathogenic budding yeast; and CaPCNA dependent DNA damage tolerance pathways could play a crucial role in addition to other cellular mechanisms like efficient drug efflux pumps, thicker cell wall and any other components present in C. albicans to maintain genomic stability.Fig. 9

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