Replication Protein
Replication protein A (RPA) is a three-subunit complex with multiple roles in DNA metabolism. DNA-binding domain A in the large subunit of human RPA (hRPA70A) binds to single-stranded DNA (ssDNA) and is responsible for the species-specific RPA-T antigen (T-ag) interaction required for Simian virus 40 replication. Although Saccharomyces cerevisiae RPA70A (scRPA70A) shares high sequence homology with hRPA70A, the two are not functionally equivalent. To elucidate the similarities and differences between these two homologous proteins, we determined the solution structure of scRPA70A, which closely resembled the structure of hRPA70A. The structure of ssDNA-bound scRPA70A, as simulated by residual dipolar coupling-based homology modeling, suggested that the positioning of the ssDNA is the same for scRPA70A and hRPA70A, although the conformational changes that occur in the two proteins upon ssDNA binding are not identical. NMR titrations of hRPA70A with T-ag showed that the T-ag binding surface is separate from the ssDNA-binding region and is more neutral than the corresponding part of scRPA70A. These differences might account for the species-specific nature of the hRPA70A-T-ag interaction. Our results provide insight into how these two homologous RPA proteins can exhibit functional differences, but still both retain their ability to bind ssDNA.
An extended DNA structure through deoxyribose-base stacking induced by RecA protein
The family of proteins that are homologous to RecA protein of Escherichia coli is essential to homologous genetic recombination in various organisms including viruses, bacteria, lower eukaryotes, and mammals. In the presence of ATP (or ATPgammaS), these proteins form helical filaments containing single-stranded DNA at the center. The single-stranded DNA bound to RecA protein is extended 1.5 times relative to B-form DNA with the same sequence, and the extension is critical to pairing with homologous double-stranded DNA. This pairing reaction, called homologous pairing, is a key reaction in homologous recombination. In this NMR study, we determined a three-dimensional structure of the single-stranded DNA bound to RecA protein. The DNA structure contains novel deoxyribose-base stacking in which the 2'-methylene moiety of each deoxyribose is placed above the base of the following residue, instead of normal stacking of adjacent bases. As a result of this deoxyribose-base stacking, bases of the single-stranded DNA are spaced out nearly 5 A. Thus, this novel structure well explains the axial extension of DNA in the RecA-filaments relative to B-form DNA and leads to a possible interpretation of the role of this extension in homologous pairing.
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