Double-check probing of DNA bending and unwinding by XPA-RPA: an architectural function in DNA repair

The multiprotein factor composed of XPA and replication protein A (RPA) is an essential subunit of the mammalian nucleotide excision repair system. Al though XPA-RPA has been implicated in damage recognition, its activity in t he DNA repair pathway remains controversial. By replacing DNA adducts with mispaired bases or non-hybridizing analogues, we found that the weak prefer ence of XPA and RPA for damaged substrates is entirely mediated by indirect readout of DNA helix conformations. Further screening with artificially di storted substrates revealed that XPA binds most efficiently to rigidly bent duplexes but not to single-stranded DNA, Conversely, RPA recognizes single -stranded sites but not backbone bending. Thus, the association of XPA with RPA generates a double-check sensor that detects, simultaneously, backbone and base pair distortion of DNA. The affinity of XPA for sharply bent dupl exes, characteristic of architectural proteins, is not compatible with a di rect function during recognition of nucleotide lesions. Instead, XPA in con junction with RPA may constitute a regulatory factor that monitors DNA bend ing and unwinding to verify the damage-specific localization of repair comp lexes or control their correct three-dimensional assembly.