Structural and Functional Characterization of Deinococcal DNA Damage Response A (DdrA)
Abstract
Deinococci exhibit a remarkable resilience toward DNA damage through the actions of several unique proteins, including DdrA. Although DdrA is critical for damage resistance, little is known about its mechanism of action. Despite sharing sequence similarity with Rad52, DdrA has been reported to lack single-stranded DNA annealing activity. In order to better characterize DdrA, structural studies were undertaken with the primary objective of gaining insight into the mechanism by which DdrA functions. Significant progress was made toward elucidating the X-ray crystal structure; in particular, identifying suitable DdrA domain boundaries for successful expression, purification and crystallization. In addition, we demonstrate for the first time that DdrA mediates ssDNA annealing to levels comparable to Rad52 in vitro. Residues (K22 and K105) critical for ssDNA binding and annealing were identified and further used to demonstrate that DdrA mediates resistance to extreme levels of DNA damage through its ability to anneal ssDNA in vivo.