Structural and functional studies on human BLM and Rad51D related in DNA repair process of homologous recombination

Abstract

The HRDC domain, conserved among members of the RecQ helicase family, regulates helicase activity by virtue of variations in its surface residues. The HRDC domain of Bloom syndrome protein (BLM) is known as a critical determinant of the dissolution function of double Holliday junctions by the BLM-TOPOIII$\alpha$ complex. In this study, we determined the solution structure of the human BLM HRDC domain and characterized its DNA binding activity. The BLM HRDC domain consists of five $\alpha$-helices with a hydrophobic $3_{10^-}$ helical loop between helices 1 and 2 and an extended acidic surface comprising residues in helices 3 to 5. The BLM HRDC domain preferentially binds to ssDNA, though with a markedly low binding affinity ($K_d$ ~ 100 $\muM$). NMR chemical shift perturbation studies suggested that the critical DNA binding residues of the BLM HRDC domain are located in the hydrophobic loop and the N-terminus of helix 2. Interestingly, the isolated BLM HRDC domain had quite different DNA binding modes between ssDNA and Holliday junctions in EMSA experiments. Based on its surface charge separation and DNA binding properties, we suggest that the HRDC domain of BLM may be adapted for a unique function among RecQ helicases-that of bridging protein and DNA interactions. The Rad51D and Xrcc2 complex, which belong to the Rad51 paralogs, are required for homologous recombinational repair (HRR) and disruption of Holliday Junction with Bloom syndrome protein (BLM) in vertebrates. The N-terminal domain of Rad51D is essential for protein-protein interaction with Xrcc2 and highly conserved in eukaryotic Rad51D orthologues, but is unknown about its individual structure or function at all. In this study, we determined the solution structure of the human Rad51D N-terminal domain (residues from 1 to 83) and characterized its DNA binding activity. The N-terminal region consists of four- short helices with a long N- and C-terminal tails. However, the N-terminal tail from Met1 ...