Autoinhibition of Bacteriophage T4 Mre11 (gp47) by its C-terminal domain [Enzymology]

July 30th, 2014 by Gao, Y., Nelson, S. W.

Mre11 and Rad50 form a stable complex (MR) and work cooperatively in repairing DNA double-strand breaks. In the bacteriophage T4, Rad50 (gp46) enhances the nuclease activity of Mre11 (gp47) and Mre11 and DNA in combination stimulate the ATPase activity of Rad50. The structural basis for the cross activation of MR complex has been elusive. Various crystal structures of MR complex display limited protein-protein interfaces that mainly exist between C-terminus of Mre11 and coiled-coil domain of Rad50. To test the role of C-terminal Rad50 binding domain (RBD) in Mre11 activation, we constructed series of C-terminal deletions and mutations in bacteriophage T4 Mre11. Deletion of the RBD in Mre11 eliminates Rad50 binding but only has moderate effect on its intrinsic nuclease activity; however, the additional deletion of highly acidic flexible linker that lies between RBD and main body of Mre11 increases the Mre11 nuclease activity by 20-fold. Replacement of the acidic residues in the flexible linker with alanine elevates the Mre11 activity to the level of MR complex when combined with deletion of RBD. Nuclease activity kinetics indicates that Rad50 association and deletion of C-terminus of Mre11 both enhance DNA substrate binding. Additionally, a short peptide that contains the flexible linker and RBD of Mre11 acts as an inhibitor of Mre11 nuclease activity. These results support a model where the Mre11 RBD and linker domain act as an autoinhibitory domain when not in complex with Rad50. Complex formation with Rad50 alleviates this inhibition due to the tight association of the RBD and the Rad50 coiled-coil.