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 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.

Structural mechanism of ATP‐dependent DNA binding and DNA end bridging by eukaryotic Rad50

“…The yeast ortholog Mre11-Rad50-Xrs2 (MRX) cleaves the 5 0 strand approximately 15-25 nt from the DNA end in vitro in a reaction that requires ATP hydrolysis, Mre11's nuclease motif, and the Sae2/CtIP protein (Garcia et al, 2011;Cannavo & Cejka, 2014;Shibata et al, 2014). Endonucleolytic cleavage of DNA near the breaks has also been observed for both archaeal and bacterial homologs, and could play a role in deprotection of DNA ends in bacteriophage T4 recombination (Connelly et al, 2003;Hopkins & Paull, 2008;Almond et al, 2013). The precise mechanism of how ATP aids and regulates nucleolytic and structural functions in all of these reactions is not understood.…”

“…In any case, by bridging DNA ends with 3 0 overhangs at microhomologies, arising after MRN-and CtIP-dependent initial processing of DNA, MRN could help to protect the DNA ends from further processing (e.g., by MRN's own ssDNA endonuclease). Since MR is important for the end-to-end coordination of recombination in bacteriophages (Almond et al, 2013), such a direct bridging of DNA ends could be a more universal function of the complex as well (Fig 7C). The DNA binding mode reported here is somewhat different to the one previously observed for the bacterial Rad50-ATP-DNA complex (Rojowska et al, 2014).…”

Functional Analysis of the Bacteriophage T4 Rad50 Homolog (gp46) Coiled-coil Domain

“…It is presumed that this end coordination is achieved through the physical tethering of the DSB ends to each other, likely through the coiledcoils of the MR complex. Depletion of T4 Rad50 results in reduced end coordination so that each end of the DSB invades different homologous templates (28,29).…”

Autoinhibition of Bacteriophage T4 Mre11 by Its C-terminal Domain

“…MR complexes in eukaryotes, archaea, and bacteriophages all catalyze DNA resection at DSBs; in contrast, the bacterial MR complex is involved in processing of cruciform structure during DNA replication (13) and DSB resection is carried out by the RecBCD helicase-nuclease complex (14). The MR complex from bacteriophage T4 functions in a similar fashion as its eukaryotic homologs and is required for both DSB repair and origin-independent DNA replication (15)(16)(17)(18).…”

“…A highly conserved CXXC motif in the middle of the coiled-coil domain forms a zinc finger-like structure with the CXXC motif from adjacent Rad50 (25). The CXXC motif is believed to mediate Rad50-dependent DNA tethering (4,18,26).…”