DNA-BINDING POLARITY OF HUMAN REPLICATION PROTEIN-A POSITIONS NUCLEASES IN NUCLEOTIDE EXCISION-REPAIR

The human single-stranded DNA-Binding replication A protein (RPA) is i nvolved in various DNA-processing events. By comparing the affinity of hRPA for artificial DNA hairpin structures with 3'- or 5'-protruding single-stranded arms, rye found that hRPA Binds ssDNA with a defined p olarity; a strong ssDNA interaction domain of hRPA is positioned at th e 5' side of its Binding region, re weak ssDNA-binding domain resides at the 3' side. Polarity appears crucial for positioning of the excisi on repair nucleases XPG and ERCC1-XPF ore the DNA. With the 3'-oriente d side of hRPA facing a duplex ssDNA junction, hRPA interacts with and stimulates ERCC1-XPF, whereas the S'-oriented side of hRPA at a DNA j unction allows stable binding of XPG to hRPA. Our data pinpoint hRPA t o the undamaged strand during nucleotide excision repair. polarity of hRPA on ssDNA is likely to contribute to the directionality of other h RPA-dependent processes as well.