title: Coordination and processing of DNA ends during double-strand break repair: the role of the bacteriophage T4 Mre11/Rad50 (MR) complex.
abstract: 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 double-strand 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.
authors: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
authors: Joshua R
authors: JR
authors: Almond
authors: Bradley A
authors: BA
authors: Stohr
authors: Anil K
authors: AK
authors: Panigrahi
authors: Dustin W
authors: DW
authors: Albrecht
authors: Scott W
authors: SW
authors: Nelson
authors: Kenneth N
authors: KN
authors: Kreuzer
mesh_terms: D017122:Bacteriophage T4 / Q000235:genetics* / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair; D045643:DNA Repair Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005814:Genes, Viral; D009154:Mutation; D011995:Recombination, Genetic; D059767:Recombinational DNA Repair; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D014764:Viral Proteins; D045643:DNA Repair Enzymes
keywords: Mre11-Rad50 complex; double-strand break (DSB) repair; end coordination; homologous recombination; recombination-dependent replication
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