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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 S. cerevisiae strains to HU and DNA damaging agents. Cells of genomic POL30 deletion yeast strains containing YEP-ADH1p-ScPCNA or YEP-ADH1p-CaPCNA from an overnight YPD culture were serially diluted and spotted onto YPD 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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Fig8: Sensitivities of S. cerevisiae strains to HU and DNA damaging agents. Cells of genomic POL30 deletion yeast strains containing YEP-ADH1p-ScPCNA or YEP-ADH1p-CaPCNA from an overnight YPD culture were serially diluted and spotted onto YPD 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: Hydroxyurea depletes cellular dNTPs level and has been widely used as a replication inhibitor. When we examined susceptibility of these strains to HU (Fig. 8A), yeast strain ectopically expressing ScPCNA (YEP-ADH1p-ScPCNA) did not show any significant sensitivity up to 50 mM HU, rather surprisingly strain expressing CaPCNA (YEP-ADH1p-CaPCNA) showed hypersensitivity to similar concentration of HU, as growth was quite adversely affected on YPD plates containing 50 mM HU. However growth at 12.5 mM HU for both the strains remained unaffected. In this assay also both the strains displayed differential growth rates and yeast strain expressing homologous PCNA was considerably over-growing the other. Although CaPCNA complemented essential nature of ScPOL30 and was able to support DNA replication, it was evident from the growth curve, temperature and HU sensitivity analyses that CaPCNA is not as efficient as ScPCNA as far as DNA replication is concerned in S. cerevisiae.Fig. 8


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 S. cerevisiae strains to HU and DNA damaging agents. Cells of genomic POL30 deletion yeast strains containing YEP-ADH1p-ScPCNA or YEP-ADH1p-CaPCNA from an overnight YPD culture were serially diluted and spotted onto YPD 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

Fig8: Sensitivities of S. cerevisiae strains to HU and DNA damaging agents. Cells of genomic POL30 deletion yeast strains containing YEP-ADH1p-ScPCNA or YEP-ADH1p-CaPCNA from an overnight YPD culture were serially diluted and spotted onto YPD 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: Hydroxyurea depletes cellular dNTPs level and has been widely used as a replication inhibitor. When we examined susceptibility of these strains to HU (Fig. 8A), yeast strain ectopically expressing ScPCNA (YEP-ADH1p-ScPCNA) did not show any significant sensitivity up to 50 mM HU, rather surprisingly strain expressing CaPCNA (YEP-ADH1p-CaPCNA) showed hypersensitivity to similar concentration of HU, as growth was quite adversely affected on YPD plates containing 50 mM HU. However growth at 12.5 mM HU for both the strains remained unaffected. In this assay also both the strains displayed differential growth rates and yeast strain expressing homologous PCNA was considerably over-growing the other. Although CaPCNA complemented essential nature of ScPOL30 and was able to support DNA replication, it was evident from the growth curve, temperature and HU sensitivity analyses that CaPCNA is not as efficient as ScPCNA as far as DNA replication is concerned in S. cerevisiae.Fig. 8

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