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 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 beta 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 strongly dependent upon the length of the template extension.
Lavrik
O I
OI
Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of the Russian Academy of Sciences. lavrik@niboch.nsc.ru
Kolpashchikov
D M
DM
Nasheuer
H P
HP
Weisshart
K
K
Favre
A
A
D003432:Cross-Linking Reagents / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry*; D006801:Humans; D019920:Photoaffinity Labels; D011487:Protein Conformation; D051716:Replication Protein A; D013698:Templates, Genetic
D003432:Cross-Linking Reagents; D017931:DNA Primers; D004268:DNA-Binding Proteins; D019920:Photoaffinity Labels; C497167:RPA1 protein, human; D051716:Replication Protein A