Functional properties of Schistosoma mansoni single-stranded DNA-binding protein SmPUR-alpha. Description of the interaction between SmPUR-alpha and SMYB1.
PUR-alpha is a highly conserved protein in eukaryotes belonging to the family of single-stranded DNA-binding proteins. Because PUR-alpha is a multifunctional protein that participates in several regulatory events at the level of gene transcription, it became relevant to investigate the structural features of Schistosoma mansoni PUR-alpha (SmPUR-alpha) that could be correlated to its mode of action. Using deletion constructs on a dot blot assay we mapped the domains of GST-SmPUR-alpha fusion protein involved in the interactions with DNA and RNA. Individually, the N-terminal amino acid residues 1-26 and the C-terminal residues 196-276 of GST-SmPUR-alpha which did not contain nucleic acid-binding domains, did not bind ssDNA or RNA. In contrast, domains encompassing the N-terminal and Class I and C-terminal plus Class I exhibited the highest binding affinity. Seemingly, the latter (GST-SmPUR-alpha 174-276) played a major role in nucleic acid interaction as judged by affinity alone. Other combinations of the deletion constructs displayed either intermediary or no binding affinity to the DNA or RNA probes. Gel shift competition assay showed that GST-SmPUR-alpha bound to ssDNA with higher affinity than to RNA. Because SmPUR-alpha contains two putative phosphorylation sites the protein was tested as a substrate to casein kinase II. GST-SmPUR-alpha could be phosphorylated in vitro by casein kinase II at both, the N- and C-terminus of the protein. The multifunctional nature of SmPUR-alpha was demonstrated by experiments measuring the physical interaction between SmPUR-alpha and the transcription factor SMYB1. This was determined in vivo (yeast two hybrid) and in vitro (GST-pull down). Furthermore, we showed that SmPUR-alpha and SMYB1 acted synergistically to bind preferentially to pyrimidine-rich sequences.
Departamento de Bioquímica Médica, ICB/CCS, Universidade Federal do Rio de Janeiro, Ilha do Fundão, Rio de Janeiro 21941-590, Brazil.
Francisco Meirelles Bastos
FM
de Oliveira
Isabel Caetano de Abreu
IC
da Silva
Franklin David
FD
Rumjanek
Analina F
AF
Valadão
Gloria R
GR
Franco
Rafael Dias
RD
Mesquita
Mario Alberto Cardoso
MA
da Silva-Neto
Marcelo Rosado
MR
Fantappié
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D047390:Casein Kinase II / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D017238:Genes, Helminth; D015801:Helminth Proteins / Q000235:genetics* / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D025941:Protein Interaction Mapping; D012313:RNA; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012550:Schistosoma mansoni / Q000502:physiology*; D017384:Sequence Deletion; D014157:Transcription Factors / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D015801:Helminth Proteins; C425295:PUR-alpha protein, Schistosoma mansoni; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C109092:Y-box-binding protein 1, Schistosoma mansoni; D012313:RNA; D047390:Casein Kinase II