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

• Published in: FEBS letters 1998-12, Vol.441 (2), p.186-190
• Main Authors: Lavrik, O.I., Kolpashchikov, D.M., Nasheuer, H.-P., Weisshart, K., Favre, A.
• Format: Article
• Language: English
• Subjects: Cross-Linking Reagents - chemistry; DNA Primers - chemistry; DNA Replication; DNA-Binding Proteins - chemistry; Human replication protein A; Humans; NAB-4-dUTP, 5-[N-(2-nitro-5-azidobenzoyl)-trans-3-aminopropenyl-1]deoxyuridine-5′-triphosphate; NAB-7-dUTP, 5-[N-[N-(2-nitro-5-azidobenzoyl)glycyl]-trans-3-aminopropenyl-1]deoxyuridine-5′-triphosphate; nt, nucleotide; PAG(E), polyacrylamide gel electrophoresis; Photoaffinity labeling; Photoaffinity Labels; Photoreactive dNTP derivative; pol β, mammalian DNA polymerase β; Protein Conformation; Protein-nucleic acid recognition; Replication Protein A; RPA, human replication protein A; SDS, sodium dodecylsulfate; ssDNA, single-stranded DNA; sTTP, 4-thiothymidine-5′-triphosphate; Templates, Genetic
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(98)01544-0

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.