Characterization of DNA-protein complex formation in nuclear extracts with a sequence from the herpes simplex virus thymidine kinase gene.
The biochemical characteristics of complex formation in nuclear extracts from mock-infected and herpes simplex virus (HSV)-infected Vero and HeLa cells with a sequence downstream of and adjacent to the promoter for the HSV thymidine kinase gene were studied using the mobility shift electrophoresis assay. This region is bound by host cell proteins, as evidenced by the formation of complexes after incubation in extracts from mock-infected cells. Unique virus-specific complexes form in extracts prepared from infected cells, and these complexes contain ICP4, the major regulatory protein of HSV. Examination of the salt requirements for assembly and the stability of preformed DNA-protein complexes to added salt demonstrate the distinct nature of the complexes that form in each extract. This finding is supported by analyses of the relative association and dissociation rates of these complexes which show that complexes formed in extracts prepared from infected cells are kinetically labile. After depletion with chelators, the divalent cation requirements for complex formation were assayed by supplementation with various metal salts. Addition of Mn2+ restored binding activity in extracts from both mock-infected and infected HeLa cells. Finally, footprinting assays revealed that sequences on each strand throughout this region of the thymidine kinase gene were involved in complex formation only in extracts from mock-infected cells. These experiments suggest that one consequence of virus gene expression is to alter the interaction of cell proteins with virus DNA.
Papavassiliou
A G
AG
Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
Silverstein
S J
SJ
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000494:pharmacology; D004587:Electrophoresis, Agar Gel; D005814:Genes, Viral; D006367:HeLa Cells; D017874:Immediate-Early Proteins; D066298:In Vitro Techniques; D007700:Kinetics; D008345:Manganese / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism*; D009994:Osmolar Concentration; D010618:Phenanthrolines / Q000494:pharmacology; D011485:Protein Binding; D012045:Regulatory Sequences, Nucleic Acid; D018139:Simplexvirus / Q000235:genetics*; D013937:Thymidine Kinase / Q000235:genetics*; D014709:Vero Cells; D054334:Viral Regulatory and Accessory Proteins / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000235:genetics*
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D009687:Nuclear Proteins; D010618:Phenanthrolines; D054334:Viral Regulatory and Accessory Proteins; D015678:Viral Structural Proteins; C048925:herpes simplex virus, type 1 protein ICP4; D008345:Manganese; D013937:Thymidine Kinase; D003850:Deoxyribonuclease I; C025205:1,10-phenanthroline