Abstract
Very little is known about protozoan replication protein A (RPA), a heterotrimeric complex critical for DNA replication and repair. We have discovered that in medically and economically important apicomplexan parasites, two unique RPA complexes may exist based on two different types of large subunit RPA1. In this study, we characterized the single-stranded DNA binding features of two distinct types (i.e. short and long) of RPA1 subunits from Cryptosporidium parvum (CpRPA1A and CpRPA1B). These two proteins differ from human RPA1 in their intrinsic single-stranded DNA binding affinity (K) and have significantly lower cooperativity (omega). We also identified the RPA2 and RPA3 subunits from C. parvum, the latter of which had yet to be reported to exist in any protozoan. Using fluorescence resonance energy transfer technology and pull-down assays, we confirmed that these two subunits interact with each other and with CpRPA1A and CpRPA1B. This suggests that the heterotrimeric structure of RPA complexes may be universally conserved from lower to higher eukaryotes. Bioinformatic analyses indicate that multiple types of RPA1 are present in the other apicomplexans Plasmodium and Toxoplasma. Apicomplexan RPA1 proteins are phylogenetically more related to plant homologues and probably arose from a single gene duplication event prior to the expansion of the apicomplexan lineage. Differential expression during the life cycle stages in three apicomplexan parasites suggests that the two RPA1 types exercise specialized biological functions.
Highlights
Cryptosporidium parvum is a unicellular pathogen that can cause persistent, potentially life-threatening watery diarrhea in both humans and animals
The ssDNA Binding of CpRPA1A and CpRPA1B Differs from That of Human RPA1—We have previously identified two different types of RPA1 subunits from C. parvum
Additional faster migrating bands were observed with both maltose-binding protein (MBP)-HsRPA1 and MBPCpRPA1B that were not observed in reactions where the products were not crosslinked to DNA [16, 33]
Summary
Preparation of Constructs—Constructs for expressing CpRPA1A, CpRPA1B, and CpRPA2 as maltose-binding protein (MBP) fusions were described previously [16, 17]. The CpRPA3 ORF was amplified with the following primers: 5Ј-CGg gat ccA TGC AGA GCT CAA TTG AAA ATG-3Ј and 5Ј-GCg aat tcT AAG CAA TAT TAG AAA CAG GCT-3Ј and engineered into the pET-24a(ϩ) vector (Novagen) at the BamHI and EcoRI sites. Each construct was maintained and amplified in Escherichia coli MachI cells (Invitrogen) grown in the presence of glucose (2 mM) to repress expression of the fusion proteins. Protein Expression and Purification—Plasmid DNA from the expression vectors described above was isolated from the MachI host cells using a QIAprep mini spin kit (Qiagen) and transferred into E. coli Rosetta cells (Novagen).
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