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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

Growth curves of various yeast strains expressing CaPCNA: a. Genomic POL30 deletion yeast strains containing YEP-ADH1p-ScPCNA (−−▲--), YCP-ADH1p-CaPCNA (−−■--) , YEP-ADH1p-CaPCNA (−−▼--), and YCP-ScPCNA gene with DE41,42AA (--●--) were grown in 100 ml YPD liquid medium at 30 °C and absorbance at 600 nm were measured at regular intervals. The plot was an average three sets of experiment. b. Total RNA from the above strains were isolated and semi-quantitive Reverse Trascriptase PCR was carried out. The PCR products were analyzed in a 1 % agarose gel electrophoresis. Lane 1, 1 KB DNA ladder; Lane 2 c-DNAs of ScPCNA DE41,42AA and ScPolη; Lane 3 c-DNAs of CaPCNA and ScPolη; Lane 4 c-DNAs of ScPCNA and ScPolη; Lane 5 c-DNAs of CaPCNA and ScPolη; Lane 6 no c-DNA control reaction.
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Fig7: Growth curves of various yeast strains expressing CaPCNA: a. Genomic POL30 deletion yeast strains containing YEP-ADH1p-ScPCNA (−−▲--), YCP-ADH1p-CaPCNA (−−■--) , YEP-ADH1p-CaPCNA (−−▼--), and YCP-ScPCNA gene with DE41,42AA (--●--) were grown in 100 ml YPD liquid medium at 30 °C and absorbance at 600 nm were measured at regular intervals. The plot was an average three sets of experiment. b. Total RNA from the above strains were isolated and semi-quantitive Reverse Trascriptase PCR was carried out. The PCR products were analyzed in a 1 % agarose gel electrophoresis. Lane 1, 1 KB DNA ladder; Lane 2 c-DNAs of ScPCNA DE41,42AA and ScPolη; Lane 3 c-DNAs of CaPCNA and ScPolη; Lane 4 c-DNAs of ScPCNA and ScPolη; Lane 5 c-DNAs of CaPCNA and ScPolη; Lane 6 no c-DNA control reaction.

Mentions: To analyze the role of CaPCNA in DNA replication, growth rates of POL30 knock out yeast strains expressing CaPCNA in single to multi-copy plasmids were monitored. Interestingly, strains harbouring CaPCNA formed smaller sized colonies and grew relatively slower than that with wild type ScPCNA, and accordingly, strain having CaPCNA in a CEN plasmid (low copy number) grew even slower (Fig. 7A, see Additional file 1: Table S1). As previously reported, we also observed a slow growth phenotype for YTS9 strain; and the slow growth phenotype was attributed to the weak binding of mutant PCNA (DE41, 42AA) with DNA and DNA polymerases [18]. Similarly, yeast strain expressing CaPCNA or ScPCNA DE41, 42AA showed temperature sensitivity at 14 °C and 35 °C (data not shown). To ascertain the slow growth defects of various yeast strains is not due to the difference in the expression levels of PCNAs rather due to the intrinsic properties of CaPCNA, RT-PCR was carried out to check the mRNA level of both the PCNA. Our result showed that there is no noticeable difference in the expression between the two PCNAs under ADH1 or under its own promoter (Fig. 7B, compare lanes 2, 4 with 3, 5). The native expression level of yeast DNA polymerase eta was checked and used as a loading control. The relative fold change of band intensities of PCNA vs ScPolη varies from ~ 0.7 to 0.9 (see Additional file 2: Table S2).Fig. 7


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)

Growth curves of various yeast strains expressing CaPCNA: a. Genomic POL30 deletion yeast strains containing YEP-ADH1p-ScPCNA (−−▲--), YCP-ADH1p-CaPCNA (−−■--) , YEP-ADH1p-CaPCNA (−−▼--), and YCP-ScPCNA gene with DE41,42AA (--●--) were grown in 100 ml YPD liquid medium at 30 °C and absorbance at 600 nm were measured at regular intervals. The plot was an average three sets of experiment. b. Total RNA from the above strains were isolated and semi-quantitive Reverse Trascriptase PCR was carried out. The PCR products were analyzed in a 1 % agarose gel electrophoresis. Lane 1, 1 KB DNA ladder; Lane 2 c-DNAs of ScPCNA DE41,42AA and ScPolη; Lane 3 c-DNAs of CaPCNA and ScPolη; Lane 4 c-DNAs of ScPCNA and ScPolη; Lane 5 c-DNAs of CaPCNA and ScPolη; Lane 6 no c-DNA control reaction.
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Fig7: Growth curves of various yeast strains expressing CaPCNA: a. Genomic POL30 deletion yeast strains containing YEP-ADH1p-ScPCNA (−−▲--), YCP-ADH1p-CaPCNA (−−■--) , YEP-ADH1p-CaPCNA (−−▼--), and YCP-ScPCNA gene with DE41,42AA (--●--) were grown in 100 ml YPD liquid medium at 30 °C and absorbance at 600 nm were measured at regular intervals. The plot was an average three sets of experiment. b. Total RNA from the above strains were isolated and semi-quantitive Reverse Trascriptase PCR was carried out. The PCR products were analyzed in a 1 % agarose gel electrophoresis. Lane 1, 1 KB DNA ladder; Lane 2 c-DNAs of ScPCNA DE41,42AA and ScPolη; Lane 3 c-DNAs of CaPCNA and ScPolη; Lane 4 c-DNAs of ScPCNA and ScPolη; Lane 5 c-DNAs of CaPCNA and ScPolη; Lane 6 no c-DNA control reaction.
Mentions: To analyze the role of CaPCNA in DNA replication, growth rates of POL30 knock out yeast strains expressing CaPCNA in single to multi-copy plasmids were monitored. Interestingly, strains harbouring CaPCNA formed smaller sized colonies and grew relatively slower than that with wild type ScPCNA, and accordingly, strain having CaPCNA in a CEN plasmid (low copy number) grew even slower (Fig. 7A, see Additional file 1: Table S1). As previously reported, we also observed a slow growth phenotype for YTS9 strain; and the slow growth phenotype was attributed to the weak binding of mutant PCNA (DE41, 42AA) with DNA and DNA polymerases [18]. Similarly, yeast strain expressing CaPCNA or ScPCNA DE41, 42AA showed temperature sensitivity at 14 °C and 35 °C (data not shown). To ascertain the slow growth defects of various yeast strains is not due to the difference in the expression levels of PCNAs rather due to the intrinsic properties of CaPCNA, RT-PCR was carried out to check the mRNA level of both the PCNA. Our result showed that there is no noticeable difference in the expression between the two PCNAs under ADH1 or under its own promoter (Fig. 7B, compare lanes 2, 4 with 3, 5). The native expression level of yeast DNA polymerase eta was checked and used as a loading control. The relative fold change of band intensities of PCNA vs ScPolη varies from ~ 0.7 to 0.9 (see Additional file 2: Table S2).Fig. 7

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