Three Isoforms of the Essential Translation Initiation Factor IF2 Differentially Modulate Homologous Recombination in Escherichia coli

In Escherichia coli, three isoforms of the essential translation initiation factor IF2 (IF2-1, IF2-2, and IF2-3) are generated from separate in-frame initiation codons in infB. The isoforms have earlier been suggested to act differentially in DNA replication restart. We report that in synthetic lethal situations associated with trapped Holliday junctions caused by deficiency of enzymes RuvAB or RuvC (that act in the post-synaptic step of homologous recombination [HR]), viability is restored in absence of any of the following: (i) IF2-1, (ii) RecA, which is the central protein for synapsis in HR, or (iii) proteins of the RecFORQ pre-synaptic HR pathway; conversely, loss of IF2-2 and IF2-3 exacerbated the synthetic defect. Strains lacking IF2-1 were also profoundly sensitive to two-ended DNA double-strand breaks (whose repair is mediated by RecA through the RecBCD pre-synaptic HR pathway), which was accompanied by reduction in extent of DNA loss around a break site. In HR assays, recovery of recombinants was diminished in IF2-1's absence. Our results suggest that isoforms IF2-1 and IF2-2/3 exert opposite effects at a step downstream of the two pre-synaptic pathways and of RecA nucleoprotein assembly, so as to increase and decrease, respectively, the efficiency of synapsis during HR

Download Full-text

Essential role for an isoform of Escherichia coli translation initiation factor IF2 in repair of two-ended DNA double-strand breaks

10.1101/2021.11.18.469101 ◽

2021 ◽

Author(s):

Jillella Mallikarjun ◽

J Gowrishankar

Keyword(s):

Escherichia Coli ◽

Translation Initiation ◽

Double Strand Breaks ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Dna Double Strand Breaks ◽

Chromosomal Locus ◽

Strand Breaks ◽

Damage Repair ◽

Replication Restart

In Escherichia coli, three isoforms of the essential translation initiation factor IF2 (IF2-1, IF2-2, and IF2-3) are generated from separate in-frame initiation codons in infB. The isoforms have earlier been suggested to additionally participate in DNA damage repair and replication restart. It is also known that the proteins RecA and RecBCD are needed for repair of DNA double-strand breaks (DSBs) in E. coli. Here we show that strains lacking IF2-1 are profoundly sensitive to two-ended DSBs in DNA generated by radiomimetic agents phleomycin or bleomycin, or by endonuclease I-SceI. However, these strains remained tolerant to other DSB-generating genotoxic agents or perturbations to which recA and recBC mutants remained sensitive, such as to mitomycin C, type-2 DNA topoisomerase inhibitors, or DSB caused by palindrome cleavage behind a replication fork. Data from genome-wide copy number analyses following I-SceI cleavage at a single chromosomal locus suggested that, in a strain lacking IF2-1, the magnitude of break induced replication through replication restart mechanisms is largely preserved but the extent of DNA resection around the DSB site is reduced. We propose that in absence of IF2-1 it is the annealing of a RecA nucleoprotein filament to its homologous target that is weakened, which in turn leads to a specific failure in assembly of Ter-to-oriC directed replisomes needed for consummation of two-ended DSB repair.

Download Full-text