Coordination and Processing of DNA Ends During Double-Strand Break Repair: The Role of the Bacteriophage T4 Mre11/Rad50 (MR) Complex

被引:11
作者
Almond, Joshua R. [1 ]
Stohr, Bradley A. [1 ]
Panigrahi, Anil K. [1 ]
Albrecht, Dustin W. [2 ]
Nelson, Scott W. [2 ]
Kreuzer, Kenneth N. [1 ]
机构
[1] Duke Univ, Med Ctr, Dept Biochem, Durham, NC 27710 USA
[2] Iowa State Univ, Dept Biochem Biophys & Mol Biol, Ames, IA 50011 USA
基金
美国国家科学基金会; 美国国家卫生研究院;
关键词
double-strand break (DSB) repair; Mre11-Rad50; complex; homologous recombination; recombination-dependent replication; end coordination; SUPPRESSOR TRANSFER-RNA; SACCHAROMYCES-CEREVISIAE; NUCLEASE ACTIVITY; ESCHERICHIA-COLI; MRE11; NUCLEASE; T4; DNA; MRE11-RAD50-XRS2; COMPLEX; RECOMBINATION PROTEIN; CRYSTAL-STRUCTURE; ATM ACTIVATION;
D O I
10.1534/genetics.113.154872
中图分类号
Q3 [遗传学];
学科分类号
071007 ; 090102 ;
摘要
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.
引用
收藏
页码:739 / 755
页数:17
相关论文
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