Figure 3.

NaBH₄ cross-linking of full-length Rev1. (A) A schematic representation of the cross-linking substrate (18-mer + ³²P-dRP) generated by pretreatment of 5′- ³²P-labeled 34-bp DNA with UDG and the expected ³²P-labeled protein DNA complex product (XL product) are shown. (B) UDG pre-treated DNA substrate (200 nM) was used in a reaction mixture without enzyme (lane 1) or with 280 and 1000 nM MBP (lanes 2 and 3) or 140, 280 and 1000 nM REV1 (lanes 4, 5, 6), or 100 nM pol β (lane 7), and 1 mM NaBH4. The reaction mixtures were incubated on ice for 1 h followed by incubation at room temperature for 10 min. Covalently cross-linked DNA-protein products were separated by 10% NuPAGE Bis-Tris gel, and the gel was scanned on a PhosphorImager. The positions of cross-linked products and free DNA are indicated. Relative positions of protein markers are indicated on the left side of the phosphorimage.