Analogues of dUTP bearing a photoreactive 2-nitro-5-azidobenzoyl (NAB) group linked via spacers of varying length (n = 2, 4, 7-13 atoms) to the 5-position of the uridine ring (NAB-n-dUTP) were synthesized and characterized. DNA polymerase beta efficiently incorporated these analogues into synthetic primer-template substrates in place of TTP, which allowed us to selectively introduce a photoreactive group at the 3' primer terminus. After completing photoreactive primer synthesis, the reaction mixtures were irradiated with monochromatic UV light (315 nm) in the presence of human replication protein A (RPA), a heterotrimer consisting of three subunits with molecular mass 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, but cross-linking of p14 was not achieved even by varying the length of the spacer 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 substrates 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 RPA and pol beta form a complex on primer-template substrates.