Synthesis of base-substituted dUTP analogues carrying a photoreactive group and their application to study human replication protein A

Analogues of dUTP bearing a photoreactive 2-nitro-5-azidobenzoyl (NAB) grou p linked via spacers of varying length (n. = 2, 4, 7-13 atoms) to the Ei-po sition of the uridine ring (NAB-n-dUTP) were synthesized and characterized. DNA polymerase beta efficiently incorporated these analogues into syntheti c primer-template substrates in place of TTP, which allowed us to selective ly introduce a photoreactive group at the 3' primer terminus. After complet ing photoreactive primer synthesis, the reaction mixtures were irradiated w ith monochromatic UV light (315 nm) in the presence of human replication pr otein A (RPA), a heterotrimer consisting of three subunits with molecular m ass 70 kDa (p70), 32 kDa (p32), and 14 kDa (p14), and were separated by SDS -PAGE. The photoreactive primers cross-linked directly with p70 and p32, bu t cross-linking of p14 was not achieved even by varying the length of the s pacer group. The data speak in favor of the protection of p14 by other RPA subunits from the interaction with 3'-end of the primer. Cross-linking of s ubstrates to pol beta is inhibited when the analogue bears a short spacer ( n = 2, 4, 7, and 8), but this is abrogated somewhat when longer spacers (n = 9-13) are examined. On the basis of these observations, we suggest that R PA and pol beta form a complex on primer-template substrates.