Limits...
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

Model structure of CaPCNA.a. Structure of CaPCNA monomer showing N- terminal domain in green, C-terminal domain in purple and IDCL in blue. b. Ring structure of CaPCNA depicting three monomers connecting N-terminal domain with C-terminal domain. c. Superimposed structures of CaPCNA (purple) and ScPCNA (green); and non-overlapping portions of CaPCNA (yellow) and ScPCNA (blue) are shown. d. The Ramachandran plot of the homology-modeled structure of CaPCNA. The red regions indicate “most favored”, yellow colored areas “additional allowed” and white portion represent “disallowed”.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: Model structure of CaPCNA.a. Structure of CaPCNA monomer showing N- terminal domain in green, C-terminal domain in purple and IDCL in blue. b. Ring structure of CaPCNA depicting three monomers connecting N-terminal domain with C-terminal domain. c. Superimposed structures of CaPCNA (purple) and ScPCNA (green); and non-overlapping portions of CaPCNA (yellow) and ScPCNA (blue) are shown. d. The Ramachandran plot of the homology-modeled structure of CaPCNA. The red regions indicate “most favored”, yellow colored areas “additional allowed” and white portion represent “disallowed”.

Mentions: Since the sliding clamps from all domains of life possess superimposable three-dimensional structures and primary sequence of CaPCNA showed remarkable similarity, we examined the structure of putative CaPCNA by using in silico approach. By using SWISS MODEL and taking ScPCNA pdb structures as template, model structure of CaPCNA was predicted (Fig. 2). CaPCNA also forms a toroidal shaped homotrimeric ring, and the superimposition of the modeled complex with the template ScPCNA showed a root mean square deviation (RMSD) of 0.48 Å (Fig. 2 B and C). The Ramachandran plot showed 88.2 % residues in the most favoured and 11.8 % residues in the additional allowed region (Fig. 2 D) confirming the acceptability of the model structure. In the monomeric structure, CaPCNA is composed of two identical globular domains, one with β1-α1-β2-β3-β4-β5-β6-α2-β7-β8-β9 and the other with β10-α3-β11-β12-β13-β14-β15-α4-β16-β17-β18, joined by an IDCL (in blue, Fig. 2 A). The minor difference in CaPCNA model structure was noticed in the loop that connects β17 and β18 at the C-terminal tail (Fig. 2 C).Fig. 2


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)

Model structure of CaPCNA.a. Structure of CaPCNA monomer showing N- terminal domain in green, C-terminal domain in purple and IDCL in blue. b. Ring structure of CaPCNA depicting three monomers connecting N-terminal domain with C-terminal domain. c. Superimposed structures of CaPCNA (purple) and ScPCNA (green); and non-overlapping portions of CaPCNA (yellow) and ScPCNA (blue) are shown. d. The Ramachandran plot of the homology-modeled structure of CaPCNA. The red regions indicate “most favored”, yellow colored areas “additional allowed” and white portion represent “disallowed”.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: Model structure of CaPCNA.a. Structure of CaPCNA monomer showing N- terminal domain in green, C-terminal domain in purple and IDCL in blue. b. Ring structure of CaPCNA depicting three monomers connecting N-terminal domain with C-terminal domain. c. Superimposed structures of CaPCNA (purple) and ScPCNA (green); and non-overlapping portions of CaPCNA (yellow) and ScPCNA (blue) are shown. d. The Ramachandran plot of the homology-modeled structure of CaPCNA. The red regions indicate “most favored”, yellow colored areas “additional allowed” and white portion represent “disallowed”.
Mentions: Since the sliding clamps from all domains of life possess superimposable three-dimensional structures and primary sequence of CaPCNA showed remarkable similarity, we examined the structure of putative CaPCNA by using in silico approach. By using SWISS MODEL and taking ScPCNA pdb structures as template, model structure of CaPCNA was predicted (Fig. 2). CaPCNA also forms a toroidal shaped homotrimeric ring, and the superimposition of the modeled complex with the template ScPCNA showed a root mean square deviation (RMSD) of 0.48 Å (Fig. 2 B and C). The Ramachandran plot showed 88.2 % residues in the most favoured and 11.8 % residues in the additional allowed region (Fig. 2 D) confirming the acceptability of the model structure. In the monomeric structure, CaPCNA is composed of two identical globular domains, one with β1-α1-β2-β3-β4-β5-β6-α2-β7-β8-β9 and the other with β10-α3-β11-β12-β13-β14-β15-α4-β16-β17-β18, joined by an IDCL (in blue, Fig. 2 A). The minor difference in CaPCNA model structure was noticed in the loop that connects β17 and β18 at the C-terminal tail (Fig. 2 C).Fig. 2

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