Functional characterization of replication protein A2 (RPA2) from Cryptosporidium parvum
- 1 May 2004
- journal article
- Published by Microbiology Society in Microbiology
- Vol. 150 (5) , 1197-1205
- https://doi.org/10.1099/mic.0.26833-0
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 parasiteCryptosporidium parvumwere characterized. Here are reported the identification and characterization of a putative middle RPA subunit (CpRPA2) from this unicellular organism. Although theCpRPA2gene 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) ofC. parvumsporozoites. 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 anin vitrophosphorylation 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 phosphorylationin vitro. These observations provide additional evidence on the divergence of RPA proteins betweenC. parvumand host, implying that the parasite DNA replication machinery could be explored as a chemotherapeutic target.Keywords
This publication has 28 references indexed in Scilit:
- Plasmodium falciparum Possesses a Cell Cycle-regulated Short Type Replication Protein A Large Subunit Encoded by an Unusual TranscriptJournal of Biological Chemistry, 2002
- Cryptosporidium virulence determinants – are we there yet?International Journal for Parasitology, 2001
- Cryptosporidium parvumpossesses a short-type replication protein A large subunit that differs from its hostFEMS Microbiology Letters, 1999
- Replication Protein A (RPA): The Eukaryotic SSBCritical Reviews in Biochemistry and Molecular Biology, 1999
- REPLICATION PROTEIN A: A Heterotrimeric, Single-Stranded DNA-Binding Protein Required for Eukaryotic DNA MetabolismAnnual Review of Biochemistry, 1997
- The DNA damage response in DNA-dependent protein kinase-deficient SCID mouse cells: Replication protein A hyperphosphorylation and p53 inductionProceedings of the National Academy of Sciences, 1996
- Functional Domains of the 70-Kilodalton Subunit of Human Replication Protein ABiochemistry, 1996
- [41] Identification of eukaryotic DNA replication proteins using simian virus 40 in Vitro replication systemPublished by Elsevier ,1995
- Isolation of the genes encoding the 51-kilodalton and 28-kilodalton subunits of Crithidia fasciculata replication protein AMolecular and Biochemical Parasitology, 1994
- Purification and characterization of replication protein A, a cellular protein required for in vitro replication of simian virus 40 DNA.Proceedings of the National Academy of Sciences, 1988