The Mre11-Rad50 complex (MR) from bacteriophage T4 (gp46/47) is involved in the processing of DNA double-strand breaks. Here, we describe the activities of the T4 MR complex and its modulation by proteins involved in homologous recombination. T4 Mre11 is a Rad50-and Mn 2؉ -dependent dsDNA exonuclease and ssDNA endonuclease. ATP hydrolysis is required for the removal of multiple nucleotides via dsDNA exonuclease activity but not for the removal of the first nucleotide or for ssDNA endonuclease activity, indicating ATP hydrolysis is only required for repetitive nucleotide removal. By itself, Rad50 is a relatively inefficient ATPase, but the presence of Mre11 and dsDNA increases ATP hydrolysis by 20-fold. The ATP hydrolysis reaction exhibits positive cooperativity with Hill coefficients ranging from 1.4 for Rad50 alone to 2.4 for the Rad50-Mre11-DNA complex. Kinetic assays suggest that approximately four nucleotides are removed per ATP hydrolyzed. Directionality assays indicate that the prevailing activity is a 3 to 5 dsDNA exonuclease, which is incompatible with the proposed role of MR in the production of 3 ssDNA ends. Interestingly, we found that in the presence of a recombination mediator protein (UvsY) and ssDNA-binding protein (gp32), Mre11 is capable of using Mg 2؉ as a cofactor for its nuclease activity. Additionally, the Mg 2؉ -dependent nuclease activity, activated by UvsY and gp32, results in the formation of endonuclease reaction products. These results suggest that gp32 and UvsY may alter divalent cation preference and facilitate the formation of a 3 ssDNA overhang, which is a necessary intermediate for recombination-mediated double-strand break repair.
Background:The Mre11 dimer interface may be dynamic, responding to ATP and/or DNA. Results: A mutation in the Mre11 dimer interface increases the nuclease initiation rate but decreases the translocation rate and lowers processivity. Conclusion: Mre11 cycles between at least two states during its catalytic cycle. Significance: The first state is for assembly/initiation and the second state is for translocation.
The in vivo functions of the bacteriophage T4 Mre11/Rad50 (MR) complex (gp46/47) in double-strand-end processing, double-strand break repair, and recombination-dependent replication were investigated. The complex is essential for T4 growth, but we wanted to investigate the in vivo function during productive infections. We therefore generated a suppressed triple amber mutant in the Rad50 subunit to substantially reduce the level of complex and thereby reduce phage growth. Growth-limiting amounts of the complex caused a concordant decrease in phage genomic recombination-dependent replication. However, the efficiencies of doublestrand break repair and of plasmid-based recombination-dependent replication remained relatively normal. Genetic analyses of linked markers indicated that double-strand ends were less protected from nuclease erosion in the depleted infection and also that end coordination during repair was compromised. We discuss models for why phage genomic recombination-dependent replication is more dependent on Mre11/Rad50 levels when compared to plasmid recombination-dependent replication. We also tested the importance of the conserved histidine residue in nuclease motif I of the T4 Mre11 protein. Substitution with multiple different amino acids (including serine) failed to support phage growth, completely blocked plasmid recombination-dependent replication, and led to the stabilization of double-strand ends. We also constructed and expressed an Mre11 mutant protein with the conserved histidine changed to serine. The mutant protein was found to be completely defective for nuclease activities, but retained the ability to bind the Rad50 subunit and double-stranded DNA. These results indicate that the nuclease activity of Mre11 is critical for phage growth and recombination-dependent replication during T4 infections.
The Rad50‐Mre11 complex (MR complex) is implicated in the initial processing of DNA double stranded breaks (DSBs). Mre11 is a nuclease and Rad50 is an ATPase belonging to the ATP Binding Cassette (ABC) protein superfamily. Rad50 forms a dimer at two interfaces: the pair of ATP binding sites that are formed in trans between two Rad50 subunits, and the zinc‐hook motif (CXXC) at the apex of an intramolecular coiled‐coil. T4 phage contains functionally equivalent orthologs of Rad50 and Mre11, gp46 and gp47 respectively. These orthologs are well expressed and easily purified from a bacterial system. The function of the zinc‐hook motif was investigated in this model system by mutating the conserved cysteines to serine (Rad502CS). The mutant was evaluated in vitro for DNA and Mre11 binding, ATP hydrolysis activity, and the activation of Mre11 nuclease activity. The results demonstrate that although Rad502CS binds Mre11 and DNA normally, the inability of the zinc‐hook to bind Zn2+ broadly affects both the ATP hydrolysis and nuclease activity of the complex. These data indicate a high degree of allosteric communication between the zinc‐hook and the active sites of both Rad50 and Mre11.
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