RPA subunit arrangement near the 3′-end of the primer is modulated by the length of the template strand and cooperative protein interactions

To analyze the interaction of human replication protein A (RPA) and its subunits with the DNA template-primer junction in the DNA replication fork, we designed several template-primer systems differing in the size of the single-stranded template tail (4, 9, 13, 14, 19 and 31 nt), Base substituted photoreactive dNTP analogs-5-[N(2-nitro-5-azidobenzoyl)-trans-amino-propenyl-1]-2'-deoxyuridine-5'-triphosphate (NAB-4-dUTP) and 5-[N-[N-(2-nitro-5-azidobenzoyl)glycyl]trans-3-aminopropenyl-1]-2'-deoxyuridine-5'-triphosphate (NAB-7-dUTP)-were used as substrates for elongation of radiolabeled primer-template by DNA polymerases in the presence or absence of RPA, Subsequent UV crosslinking sl-towed that the pattern of p32 and p70 RPA subunit labeling, and consequently their interaction with the template-primer junction, is strongly dependent on the template extension length at a particular RPA concentration, as well as on the ratio of RPA to template concentration. Our results suggest a model of changes in the RPA configuration modulating by the length of the template extension in the course of nascent DNA synthesis.