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Volume 150, Issue 5

Functional characterization of replication protein A2 (RPA2) from Free

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 were characterized. Here are reported the identification and characterization of a putative middle RPA subunit (CpRPA2) from this unicellular organism. Although the 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 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 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 . These observations provide additional evidence on the divergence of RPA proteins between and host, implying that the parasite DNA replication machinery could be explored as a chemotherapeutic target.

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Keyword(s): DNA-PK, DNA-dependent protein kinase , EMSA, electrophoretic mobility shifting assay , MBP, maltose-binding protein , rCpRPA2, recombinant CpRPA2 protein and RPA, replication protein A
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2004-05-01
2024-04-20
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Functional characterization of replication protein A2 (RPA2) from Cryptosporidium parvum
Microbiology 150, 1197 (2004); https://doi.org/10.1099/mic.0.26833-0
/content/journal/micro/10.1099/mic.0.26833-0
/content/journal/micro/10.1099/mic.0.26833-0
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