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)-frans-3-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′-triphos-phate (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 showed 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.