Localization of adjacent binding domains for cellular proteins over the minute virus of mice P4 promoter by site-specific photoaffinity labelling.

A photoaffinity labelling (PHL) procedure was used to localize the specific binding sites for A92L fibroblast nuclear proteins on the minute virus of mice (MVM) P4 promoter. We describe a chemical and biochemical method for the construction of precisely modified photoreactive (phr) DNA probes. In this method, a phenylazide group is attached to the DNA fragment by coupling SASD [sulfosuccinimidyl-2-(p-azidosalicyl-amido)ethyl-1,3'-dithiopro pio nate] to the primary amino group of the linker arm present at any predetermined position. These phr probes would identify, upon photocrosslinking, only those proteins which bind to the location of the phr group. Specifically, two phr probes representing the 139-172 bp region of the MVM P4 promoter were constructed in which the highly phr phenylazide group was attached with a linker at nucleotide 168, towards the side of the GC box proximal to the TATA box. The PHL studies with these photoprobes revealed that although the proteins of 95 and 120-kDa bind near nt 168 of the P4 promoter, the 120-kDa protein requires the region between the TATA box and the GC box for binding to the MVM P4 promoter.