| The recently published crystal structure of RecBCD enzyme complexed with a DNA hairpin revealed several striking structures: a putative "chi-scanning site" in RecC, an "arm" in RecB and a "pin" in RecC. I applied site-directed mutagenesis to identify their functions.;First, I established the important residues responsible for chi recognition in the channel passed through by the 3'-ended ssDNA in RecC. Residues in the channel were mutagenized to alanines individually and three types of mutants were identified based on genetic screening. Here, biochemically, the "Lost-recognition" mutants lose the ability to respond to chi. The "Relaxed-specificity" and "Reduced-specificity" mutants still recognize chi; moreover, they are able to recognize some single base mutants of chi with different efficiencies. These findings provide new information on how RecBCD enzyme recognizes and interacts with the chi sequence.;Second, I determined the role of the "arm" structure in RecB that contacts the incoming duplex DNA. When the "arm" structure was deleted, the resultant recBarm-CD cells are UV resistant while the recBarm-C cells are sensitive to UV irradiation. Consistently, in vitro, the RecBarm- CD holoenzyme is fully functional, displaying wild-type level helicase and nuclease activities, although the affinity for duplex DNA ends is lower than RecBCD. Moreover, RecBarm-CD enzyme could respond to chi as RecBCD does, suggesting the "armless" RecB is active during translocation. However, the affinity of RecBarm-C protein for dsDNA ends is dramatically lower than RecBC protein. These results suggest that the "arm" plays a direct role in dsDNA binding.;Third, the methionine "pin" in RecC at the junction between ssDNA and dsDNA was studied. The methionine dyad was replaced with two alanines. The recBCpin-D cells are UV resistant in vivo. Consistently, purified RecBCpin-D protein possesses wild-type level of dsDNA-dependent ATPase, helicase and nuclease activities, as well as chi response. The implications of these results on the proposed function of the "pin" are discussed.;Fourth, I studied the translocation polarity of RecBCD enzyme. The nuclease deficient mutant RecBD1080ACD enzyme could bypass gaps on either strand of the duplex DNA, suggesting that RecBCD translocates on both strands of DNA. |
