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

Abstract
To analyze the influence of single‐stranded template extension of DNA duplex 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 single‐stranded template tail (9 and 19 nucleotides (nt)). Base‐substituted photoreactive dUTP analogs were used as substrates for elongation of radiolabeled template‐primer by DNA polymerase β in the absence or in the presence of RPA. Following UV‐crosslinking it was demonstrated that the pattern of RPA subunit labeling and consequently RPA arrangement near the 3′‐end of the primer is stronlgly dependent upon the length of the template extension.