Alternative conformations of human replication protein A are detected by crosslinks with primers carrying a photoreactive group at the 3 '-end

To analyze the influence of single-stranded template extension of DNA duple x on the conformation of human replication protein A (RPA) bound to DNA we have designed two template-primer systems differing by the size of the sing le-stranded template tail (9 and 19 nucleotides (nt)), Base-substituted pho toreactive dUTP analogs were used as substrates for elongation of radiolabe led template-primer by DNA polymerase beta in the absence or in the presenc e of RPA, Following UV-crosslinking it was demonstrated that the pattern of RPA subunit labeling and consequently RPA arrangement near the 3'-end of t he primer is stronlgly dependent upon the length of the template extension. (C) 1998 Federation of European Biochemical Societies.