Publicação: RPA-1 from Leishmania sp.: Recombinant Protein Expression and Purification, Molecular Modeling, and Molecular Dynamics Simulations Protocols
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RPA is a conserved heterotrimeric complex and the major single-stranded DNA (ssDNA)-binding protein heterotrimeric complex, which in eukaryotes is formed by the RPA-1, RPA-2, and RPA-3 subunits. The main structural feature of RPA is the presence of the oligonucleotide/oligosaccharide-binding fold (OB-fold) domains, responsible for ssDNA binding and protein:protein interactions. Among the RPA subunits, RPA-1 bears three of the four OB folds involved with RPA-ssDNA binding, although in some organisms RPA-2 can also bind ssDNA. The OB-fold domains are also present in telomere end-binding proteins (TEBP), essential for chromosome end protection. RPA-1 from Leishmania sp., as well as RPA-1 from trypanosomatids, a group of early-divergent protozoa, shows some structural differences compared to higher eukaryote RPA-1. Also, RPA-1 from Leishmania sp., similar to TEBPs, may exert telomeric protective functions. Remarkably, different pieces of evidence have pointed out that trypanosomatids may not have OB fold-containing TEBPs. Moreover, recent data indicate that trypanosomatid RPA-1 may be considered a TEBP since it shares with TEBPs conserved functional and structural features. However, it is still unknown whether the RPA-1 protective telomeric role is exclusive to trypanosomatids or is also present in other primitive eukaryotes. Here, we describe a protocol to obtain highly purified and biologically active Leishmania amazonensis recombinant RPA-1, and to perform molecular modeling and molecular dynamics simulations methods which could be probably applied to functional and structural studies of homologous proteins in other primitive eukaryotes.
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Leishmania, OB-fold, RPA, TEBP, Telomeres, Trypanosomatids
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Inglês
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Methods in Molecular Biology, v. 2281, p. 169-191.
