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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

Physical interaction of CaPCNA with ScPolη: About 25-500nM of ScPolη was injected on CaPCNA or ScPCNA or BSA immobilized GLC chip as indicated with running buffer at a flow rate of 50 μl/min for 180 s with a 600 s dissociation phase. The dissociation constants were determined.
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Fig5: Physical interaction of CaPCNA with ScPolη: About 25-500nM of ScPolη was injected on CaPCNA or ScPCNA or BSA immobilized GLC chip as indicated with running buffer at a flow rate of 50 μl/min for 180 s with a 600 s dissociation phase. The dissociation constants were determined.

Mentions: PCNA functions as a docking site for DNA polymerases and other proteins during various processes of DNA transaction. In order to function, CaPCNA needs to interact with DNA polymerases and other associated proteins. Yeast DNA polymerase eta (ScPolη) that takes part in translesion DNA synthesis [35] was used as a candidate polymerase for physical interaction study by surface plasmon resonance. We immobilized CaPCNA or ScPCNA on a GLC- chip to determine the binding affinity for ScPolη. As a control, required amount of BSA was also immobilized. When ScPolη was passed over BSA, we did not see any significant increase in response unit where as it showed ~250 and ~650 RU for ScPCNA and CaPCNA, as ligands respectively (Fig. 5) suggesting the tight binding between PCNAs and DNA polymerase. The rate of dissociation of ScPolη from ScPCNA surface (kd:1.38 x 10−3 s-1) was comparable to that from CaPCNA immobilized surface (kd:1.66 x 10−4 s-1). The KD values of CaPCNA and ScPCNA for ScPolη were determined as 10.6 nM and 80.13 nM, respectively.Fig. 5


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)

Physical interaction of CaPCNA with ScPolη: About 25-500nM of ScPolη was injected on CaPCNA or ScPCNA or BSA immobilized GLC chip as indicated with running buffer at a flow rate of 50 μl/min for 180 s with a 600 s dissociation phase. The dissociation constants were determined.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: Physical interaction of CaPCNA with ScPolη: About 25-500nM of ScPolη was injected on CaPCNA or ScPCNA or BSA immobilized GLC chip as indicated with running buffer at a flow rate of 50 μl/min for 180 s with a 600 s dissociation phase. The dissociation constants were determined.
Mentions: PCNA functions as a docking site for DNA polymerases and other proteins during various processes of DNA transaction. In order to function, CaPCNA needs to interact with DNA polymerases and other associated proteins. Yeast DNA polymerase eta (ScPolη) that takes part in translesion DNA synthesis [35] was used as a candidate polymerase for physical interaction study by surface plasmon resonance. We immobilized CaPCNA or ScPCNA on a GLC- chip to determine the binding affinity for ScPolη. As a control, required amount of BSA was also immobilized. When ScPolη was passed over BSA, we did not see any significant increase in response unit where as it showed ~250 and ~650 RU for ScPCNA and CaPCNA, as ligands respectively (Fig. 5) suggesting the tight binding between PCNAs and DNA polymerase. The rate of dissociation of ScPolη from ScPCNA surface (kd:1.38 x 10−3 s-1) was comparable to that from CaPCNA immobilized surface (kd:1.66 x 10−4 s-1). The KD values of CaPCNA and ScPCNA for ScPolη were determined as 10.6 nM and 80.13 nM, respectively.Fig. 5

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