Identification of proteins interacting with newly replicated DNA in SV40-infected cells by UV-induced DNA-protein cross-linking.
Protein species interacting with newly replicated DNA were analyzed using a photo cross-linking technique. Nascent DNA was labeled in vitro with [alpha-32P]dCTP and BrdUTP in SV40-infected CV-1 cells made permeable with saponin. The labeled cells were then irradiated with UV light (254 nm) and were treated extensively with DNase I. Proteins with radioactive DNA tags were separated by SDS-PAGE and visualized by autoradiography. Among 10-15 proteins which were cross-linked, the proteins with apparent molecular weights of 16.5 K, 44 K, 82 K and those in the 94-140 K region appeared to be associated with newly replicated SV40 DNA. A pulse-chase experiment showed that the 82 K and 94-140 K proteins interacted with new DNA in a relatively localized region close to the replication fork. The 44 K protein was identified as the major viral capsid protein, VP1, using antiserum to SV40 capsid proteins. It was suggested that VP1 binds to nascent DNA shortly after DNA synthesis and migrates into chromatin maturation regions.
D000818:Animals; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000528:radiation effects*; D002522:Chlorocebus aethiops; D003432:Cross-Linking Reagents; D004261:DNA Replication / Q000528:radiation effects*; D004279:DNA, Viral / Q000235:genetics*; D003851:Deoxyribonucleases; D007668:Kidney; D007700:Kinetics; D008970:Molecular Weight; D013539:Simian virus 40 / Q000235:genetics*; D014466:Ultraviolet Rays; D014764:Viral Proteins / Q000235:genetics* / Q000302:isolation & purification
D003432:Cross-Linking Reagents; D004279:DNA, Viral; D014764:Viral Proteins; D003851:Deoxyribonucleases