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J. Biol. Chem., Vol. 280, Issue 36, 31460-31469, September 9, 2005

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The Protozoan Parasite Cryptosporidium parvum Possesses Two Functionally and Evolutionarily Divergent Replication Protein A Large Subunits^*

S. Dean Rider, Jr., Xiaomin Cai, William J. Sullivan, Jr., Aaron T. Smith, Jay Radke¶, Michael White¶, and Guan Zhu||**

From the Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences and the ^||Faculty of Genetics Program, Texas A&M University, 4467 TAMU, College Station, Texas 77843, the Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana 46202, and the ^¶Department of Veterinary Molecular Biology, Montana State University, Bozeman, Montana 59717

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 (). 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.

Received for publication, April 25, 2005 , and in revised form, June 24, 2005.

^* This work was supported by National Institutes of Health Grant R21 AI055278 (to G. Z.). This work was also supported in part by American Heart Association Grant 0235424Z (to W. J. S.).

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY994152.

The on-line version of this article (available at http://www.jbc.org) contains supplemental information on T. gondii RPA predicted proteins.

^** To whom correspondence should be addressed: Dept. of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A & M University, 4467 TAMU, College Station, TX 77843. Tel.: 979-865-6981; Fax: 979-845-9972; E-mail: gzhu{at}cvm.tamu.edu.

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