Binding of XPA and RPA to damaged DNA investigated by fluorescence anisotrophy

The proteins XPA and RPA are assumed to be involved in primary damage recog nition of global genome nucleotide excision repair. XPA as well as RPA have been each reported to specifically bind DNA lesions, and ternary complex f ormation with damaged DNA has also been shown. We employed fluorescence ani sotropy measurements to study the DNA-binding properties of XPA and RPA und er true equilibrium conditions using damaged DNA probes carrying at termina l fluorescein modification as a reporter. XPA binds with low affinity and i n a strongly salt-dependent manner to DNA containing a 1,3-d(GTG) intrastra nd adduct of the anticancer drug cisplatin or a 6-nt mismatch (K-D = 400 nM ) with 3-fold preference for damaged vs undamaged DNA. At near physiologica l salt conditions binding is very weak (K-D > 2 muM). RPA binds to damaged DNA probes with dissociation constants in the range of 20 nM and a nearly 1 5-fold preference over undamaged DNA. The presence of a cisplatin modificat ion weakens the affinity of RPA for single-stranded DNA by more than 1 orde r of magnitude indicating that binding to the lesion itself is not a drivin g force in damage recognition. Our fluorescence anisotropy assays also show that the presence of XPA does not enhance the affinity of RPA for damaged DNA although both proteins interact. In contrast, cooperative binding of XP A and RPA is observed in EMSA. Our results point to a damage-sensing functi on of the XPA-RPA complex with RPA mediating the important DNA contacts.