Abstract
Homologous recombination (HR) plays an essential role in the maintenance of genome integrity. RecA/Rad51 paralogs have been recognized as an important factor of HR. Among them, only one bacterial RecA/Rad51 paralog, RadA, is involved in HR as an accessory factor of RecA recombinase. RadA has a unique Lon protease-like domain (LonC) at its C terminus, in addition to a RecA-like ATPase domain. Unlike Lon protease, RadA's LonC domain does not show protease activity but is still essential for RadA-mediated DNA repair. Reconciling these two facts has been difficult because RadA's tertiary structure and molecular function are unknown. Here, we describe the hexameric ring structure of RadA's LonC domain, as determined by X-ray crystallography. The structure revealed the two positively charged regions unique to the LonC domain of RadA are located at the intersubunit cleft and the central hole of a hexameric ring. Surprisingly, a functional domain analysis demonstrated the LonC domain of RadA binds DNA, with site-directed mutagenesis showing that the two positively charged regions are critical for this DNA-binding activity. Interestingly, only the intersubunit cleft was required for the DNA-dependent stimulation of ATPase activity of RadA, and at least the central hole was essential for DNA repair function. Our data provide the structural and functional features of the LonC domain and their function in RadA-mediated DNA repair.
Highlights
Homologous recombination (HR) plays an essential role in the maintenance of genome integrity
To verify the involvement of RadA in DNA repair, the phenotype of ⌬radA was characterized by measuring the sensitivity to UV light and mitomycin C, which introduce DNA strand breaks via DNA adducts and cross-links (Fig. 5C). ⌬radA was ϳ30-fold more sensitive than the wild type to both UV and mitomycin C treatments. These results imply that RadA functions as a DNA-repair protein in T. thermophilus HB8 as it does in other organisms, such as E. coli and D. radiodurans [16, 22]
We further revealed that rLonC possesses DNA-binding activity, which is exerted by two positively charged regions, namely the intersubunit cleft and central hole of the ring (Figs. 1, 3, and 4)
Summary
Homologous recombination (HR) plays an essential role in the maintenance of genome integrity. Novel Lon protease-like DNA-binding domain of RecA paralog itively charged regions, namely the intersubunit cleft and the central hole of the hexameric ring structure. We showed that these two DNA-binding sites have different roles in the DNA-dependent ATPase activity of RadA in vitro and that at least the central hole is essential to the RadA-mediated DNA repair function in vivo. Combining these results with a phylogenetic analysis highlighted structural and functional relationships between RadA and another LonC family protein, ComM. Our findings provide the structural and functional aspects of the rLonC domain in RadA-mediated DNA repair and evolutionary aspects of the LonC family proteins
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