THE RPA32 SUBUNIT OF HUMAN REPLICATION PROTEIN-A CONTAINS A SINGLE-STRANDED DNA-BINDING DOMAIN

Replication protein A (RPA) is a conserved nuclear single-stranded DMA (ssDNA)-binding protein, Human RPA (hRPA) comprises three subunits of approximately 70, 32, and 14 kDa (hRPA70, hRPA32 and hRPA14), RPA is known to bind ssDNA through two ssDNA-binding domains in the RPA70 sub unit. Here, we demonstrate that the complex of hRPA32 and hRPA14 has a n ssDNA-binding domain, Limited proteolysis of the hRPA14.32 complex d efined a cars dimes composed of the central region of hRPA32 (amino ac ids 43-171) and RPA14. The core dimes bound ssDNA with an affinity of approximately 10-50 mu M, which is at least 100-fold more avid than th e DNA-binding affinity of the intact dimer. Analysis of the predicted secondary structure of hRPA32 suggests that amino acids 63-150 of hRPA 32 form an ssDNA-binding domain similar in structure to each of those in hRPA70, The complex of hRPA14 and hRPA32-(43-171) in turn formed a trimeric complex with the C-terminal region of hRPA70 (amino acids 436 -616), The ssDNA-binding affinity of this trimeric complex was 3 to 5- fold higher than hRPA14.32-(43-171) alone, suggesting a role far the C terminus of hRPA70 in ssDNA binding.