E. Bochkareva et al., THE RPA32 SUBUNIT OF HUMAN REPLICATION PROTEIN-A CONTAINS A SINGLE-STRANDED DNA-BINDING DOMAIN, The Journal of biological chemistry, 273(7), 1998, pp. 3932-3936
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.