Replication protein A interactions with DNA. III. Molecular basis of recognition of damaged DNA

Human replication protein A (RPA) is a heterotrimeric single-stranded DNA-b inding protein (subunits of 70, 32, and 14 kDa) that is required for cellul ar DNA metabolism, RPA has been reported to interact specifically with dama ged double-stranded DNA and to participate in multiple steps of nucleotide excision repair (NER) including the damage recognition step. We have examin ed the mechanism of RPA binding to both single-stranded and double-stranded DNA (ssDNA and dsDNA, respectively) containing damage. We show that the af finity of RPA for damaged dsDNA correlated with disruption of the double he lix by the damaged bases and required RPAs ssDNA-binding activity. We concl ude that RPA is recognizing single-stranded character caused by the damaged nucleotides. We also show that RPA binds specifically to damaged ssDNA. Th e specificity of binding varies with the type of damage with RPA having up to a 60-fold preference for a pyrimidine(6-4)pyrimidone photoproduct. We sh ow that this specific binding was absolutely dependent on the zinc-finger d omain in the C-terminus of the 70-kDa subunit. The affinity of RPA for dama ged ssDNA was 5 orders of magnitude higher than that of the damage recognit ion protein XPA (xeroderma pigmentosum group A protein). These findings sug gest that RPA probably binds to both damaged and undamaged strands in the N ER excision complex. RPA binding may be important for efficient excision of damaged DNA in NER.