Back to Search
Start Over
Functional characterization of replication protein A2 (RPA2) from Cryptosporidium parvum.
- Source :
-
Microbiology (Reading, England) [Microbiology (Reading)] 2004 May; Vol. 150 (Pt 5), pp. 1197-1205. - Publication Year :
- 2004
-
Abstract
- Replication protein A (RPA) is a heterotrimeric complex of single-stranded DNA-binding proteins that play multiple roles in eukaryotic DNA metabolism. The RPA complex is typically composed of heterologous proteins (termed RPA1, RPA2 and RPA3) in animals, plants and fungi, which possess different functions. Previously, two distinct, short-type RPA large subunits (CpRPA1 and CpRPA1B) from the apicomplexan parasite Cryptosporidium parvum were characterized. Here are reported the identification and characterization of a putative middle RPA subunit (CpRPA2) from this unicellular organism. Although the CpRPA2 gene encodes a predicted 40.1 kDa peptide, which is larger than other RPA2 subunits characterized to date, Western blot analysis of oocyst preparations detected a native CpRPA2 protein with a molecular mass of approximately 32 kDa, suggesting that CpRPA2 might undergo post-translational cleavage or the gene was translated at an alternative start codon. Immunofluorescence microscopy using a rabbit anti-CpRPA2 antibody revealed that CpRPA2 protein was mainly distributed in the cytosol (rather than the nuclei) of C. parvum sporozoites. Semi-quantitative RT-PCR data indicated that CpRPA2 was differentially expressed in a tissue culture model with highest expression in intracellular parasites infecting HCT-8 cells for 36 and 60 h. Sequence comparison suggests that RPA2 is a group of poorly conserved proteins. Nonetheless, functional analyses of recombinant proteins confirmed that CpRPA2 is a single-stranded DNA-binding protein and that it could serve as an in vitro phosphorylation target by a DNA-dependent protein kinase. The minimal length of poly(dT) required for CpRPA2 binding is 17 nucleotides, and the DNA-binding capability was inhibited by phosphorylation in vitro. These observations provide additional evidence on the divergence of RPA proteins between C. parvum and host, implying that the parasite DNA replication machinery could be explored as a chemotherapeutic target.
- Subjects :
- Amino Acid Sequence
Animals
Blotting, Western
Cell Line
Cryptosporidium parvum genetics
Cryptosporidium parvum growth & development
Cytosol metabolism
DNA, Single-Stranded metabolism
DNA-Binding Proteins chemistry
DNA-Binding Proteins genetics
Microscopy, Fluorescence
Molecular Sequence Data
Protozoan Proteins chemistry
Protozoan Proteins genetics
Protozoan Proteins metabolism
Rabbits
Replication Protein A
Sequence Analysis, DNA
Sporozoites genetics
Sporozoites growth & development
Cryptosporidium parvum metabolism
DNA Replication
DNA-Binding Proteins metabolism
Sporozoites metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1350-0872
- Volume :
- 150
- Issue :
- Pt 5
- Database :
- MEDLINE
- Journal :
- Microbiology (Reading, England)
- Publication Type :
- Academic Journal
- Accession number :
- 15133081
- Full Text :
- https://doi.org/10.1099/mic.0.26833-0