Relationship of oligomerization to DNA binding of Wheat dwarf virus RepA and Rep proteins.
Members of the genus Mastrevirus (family Geminiviridae) produce a complementary-sense (c-sense) transcription unit with the potential to encode two proteins, RepA and Rep. In the present work, we have studied the DNA-protein complexes formed by the Wheat dwarf virus (WDV) RepA protein within the WDV large intergenic region. WDV RepA forms large nucleoprotein complexes near the TATA boxes of the viral complementary-sense and virion-sense (v-sense) promoters (the RepA C- and V-complexes, respectively), a location similar to those of WDV Rep-DNA complexes but with distinct DNase I footprints. We have also studied the relationship of oligomerization of WDV RepA and Rep proteins to DNA-protein complex formation. Using chemical cross-linking, we have determined that both WDV proteins can form oligomers in solution. Interestingly, the pH is critical for the monomer-oligomer equilibrium and small changes produce a displacement in such a way that at pH </= 7.0, the predominant species is an octamer while at pH >/= 7.4 it is a monomer. Complex formation is also strongly affected by pH and occurs more efficiently at pH 7.0-7.4. We found that preformed oligomers interact very poorly with DNA. Thus, our data are consistent with a stepwise model for protein-DNA complex assembly in which monomers interact with DNA and then with other monomers to assemble an oligomeric structure on the DNA. These results may be relevant for studies on the DNA binding, replication, and transcription properties of geminivirus proteins.
Missich
R
R
Centro de Biología Molecular "Severo Ochoa,", Consejo Superior de Investigaciones Cientificas (CSIC)-Universidad Autónoma de Madrid (UAM), Cantoblanco, Madrid, 28049, Spain.
Ramirez-Parra
E
E
Gutierrez
C
C
D001483:Base Sequence; D003432:Cross-Linking Reagents; D018983:DNA Footprinting; D004265:DNA Helicases; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D017791:Geminiviridae / Q000737:chemistry / Q000235:genetics; D005814:Genes, Viral / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D012996:Solutions; D016385:TATA Box / Q000235:genetics; D013816:Thermodynamics; D015534:Trans-Activators; D014908:Triticum / Q000821:virology; D020798:Two-Hybrid System Techniques; D014764:Viral Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D003432:Cross-Linking Reagents; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D011506:Proteins; D012996:Solutions; D015534:Trans-Activators; D014764:Viral Proteins; C045264:replication initiator protein; D003850:Deoxyribonuclease I; D004265:DNA Helicases