Essential Role for an Isoform of Escherichia coli Translation Initiation Factor IF2 in Repair of Two-Ended DNA Double-Strand Breaks

Abstract

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 recombination-dependent replication through replication restart mechanisms is largely preserved but the extent of DNA resection around the DSB site is reduced. We propose that in the absence of IF2-1 it is the synapsis 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. IMPORTANCE Double-strand breaks (DSBs) in DNA are major threats to genome integrity. In Escherichia coli, DSBs are repaired by RecA- and RecBCD-mediated homologous recombination (HR). This study demonstrates a critical role for an isoform (IF2-1) of the translation initiation factor IF2 in the repair of two-ended DSBs in E. coli (that can be generated by ionizing radiation, certain DNA-damaging chemicals, or endonuclease action). It is proposed that IF2-1 acts to facilitate the function of RecA in the synapsis between a pair of DNA molecules during HR.

Keywords: DNA repair; double-strand breaks; translation initiation factor IF2.

FIG 1

Demonstration by dilution-spotting experiments of sensitivity to two-ended DSBs in DNA in the absence of IF2-1. In the different panels, strains designated IF2(wt), ΔIF2-1, or ΔIF2-2,3 were also ΔinfB at the native chromosomal locus. Growth medium was LB with supplements as indicated on top; for the genotoxic agents, numbers in parentheses refer to concentrations in μg/mL, while Glu and Ara were each at (0.2%). Relevant strain genotypes/features are shown at left (strains of panel D carried the construct for Ara-inducible expression of I-SceI and the cognate cut site). Strains employed for different rows were (from top, all strain numbers are prefixed with GJ): panel A – 19844, 19193, 19194, 15494, 15495, and 19812; panel B - 15494 and 15494/pHYD5212; panel C - 15410, 19816, and 19817; and panel D – 15837, 19804, 19805, 19806, and 19818.

FIG 2

Demonstration by flow cytometry of sensitivity to two-ended DSBs in DNA in the absence of IF2-1. Flow cytometry was performed following propidium iodide staining of cells in LB-grown cultures of strains whose relevant genotypes/features are indicated on top and perturbations, if any, at left; strains designated IF2(wt), ΔIF2-2,3, or ΔIF2-1 were also ΔinfB. Phleo supplementation was at 2 μg/mL, and I-SceI refers to Ara-supplementation in cultures of derivatives carrying P_ara::I-SceI and the cognate cut site in lacZ. In each subpanel, the percentage of total cells whose intensity of propidium iodide staining exceeded the threshold that was taken to demarcate dead cells (10³ arbitrary units, marked by vertical line), is indicated at top right. Strains employed were (all strain numbers are prefixed with GJ): I. derivatives without P_ara::I-SceI – IF2(wt), 19193; ΔIF2-2,3, 19194; ΔIF2-1, 15494; recA, 19844; and II. derivatives with P_ara::I-SceI – IF2(wt), 19804; ΔIF2-2,3, 19805; ΔIF2-1, 19806; and recA, 19807.

FIG 3

Tolerance of strains expressing different IF2 isoforms to other genotoxic agents and perturbations. Dilution-spotting assays were performed on LB with supplements as indicated on top; numbers in parentheses refer to concentrations in μg/mL. Cipro, ciprofloxacin; Nal, nalidixic acid; and MMC, mitomycin C. Relevant strain genotypes are shown at left. Strains whose designations include IF2(wt), ΔIF2-1, or ΔIF2-2,3 were also ΔinfB. In the strains of panel B, SbcCD expression that is induced in cells on Ara-supplemented medium leads to cleavage of the sister chromatid associated with the lagging-strand template at an engineered palindrome sequence in the lacZ gene (41). Strains used were (from top, unless otherwise indicated all strain numbers mentioned are prefixed with GJ): in panel A – 19844, 19193, 19194, 15494, 15495, and 19812; and in panel B – DL2006, 19811, 19808, 19809, and 19810.

FIG 4

Overexpression of isoforms IF2-2,3 or IF2-3 confers sensitivity to two-ended DSBs in DNA. Dilution-spotting assays were performed on LB with Amp and IPTG, along with the additional supplements as indicated on top; numbers in parentheses refer to concentrations in μg/mL. Nal, nalidixic acid. Relevant strain genotypes are shown at left (all were ΔinfB). Strains used for different rows were derivatives of GJ19193 with following plasmids (from top): vector pTrc99A, pHYD5207, and pHYD5208.

FIG 5

Chromosomal DNA copy number analysis by WGS in strains expressing different IF2 isoforms, following two-ended DSB generation at lacZ. DNA copy numbers (after normalization) are plotted as semilog graphs for overlapping 10-kb intervals across the genome for derivatives each carrying P_ara::I-SceI and the cognate cut site in lacZ, after supplementation of cultures grown in LB with 0.2% Glu (control) or Ara for 1 h (top and bottom rows, respectively). Relevant genotypes are indicated at top of each pair of panels; all strains were also ΔinfB. In these Cartesian graphical representations, the circular 4642-kb long chromosome is shown linearized at oriC, with genome coordinates on the abscissa corresponding to the MG1655 reference sequence (wherein oriC is at 3926 kb). Ordinate scales (log₂) shown at left on top and bottom rows are common for, respectively, panels i-iv and v-vii. The positions of lacZ, TerA and TerC/B are marked. Strains used were (all strain numbers are prefixed with GJ): IF2(wt), 19804; ΔIF2-2,3, 19805; ΔIF2-1, 19806; and IF2(wt) recA, 19818.

FIG 6

Effects of rec (A) or pri (B) mutations on DNA copy numbers in IF2(wt) or ΔIF2-1 strains following two-ended DSB generation at lacZ. Each derivative carried P_ara::I-SceI and the cognate cut site in lacZ, and cultures grown in LB were supplemented with Ara for 1 h. Representations of WGS analysis and notations used are as described in legend to Fig. 5. Ordinate scales (log₂) shown at left are common for all subpanels in that row. Strains used for the different subpanels were (all strain numbers are prefixed with GJ): (A) i, 19857; ii, 19861; and iii, 19863; and (B) i, 19864; ii, 19858; iii, 19867; iv, 19866; v, 19860; and vi, 19869.

FIG 7

Suppression by rpoB*35 mutation of I-SceI cleavage lethality in ΔIF2-1 strain. Dilution-spotting assays, of strains carrying the construct for Ara-inducible expression of I-SceI and the cognate cut site, were performed on LB supplemented with Glu or Ara (each at 0.2%), as indicated. Relevant strain genotypes/features are shown at left. Note that the growth observed for the strains recA and ΔIF2-1 on Ara-supplemented medium is that of suppressor mutants. Strains employed for different rows were (from top, all strain numbers are prefixed with GJ): 19891, 19818, 19896, 19852, 19892, and 19897.

FIG 8

Model to explain failure of two-ended DSB repair in the absence of IF2-1. (A) In IF2(wt) strain, cleavage at I-SceI site in lacZ (red arrow) leads to DNA resection by RecBCD of the two ends toward oriC and Ter, respectively (interrupted lines), followed by two events of RecA-mediated strand invasion into a sister DNA duplex (subpanel 1). “Ends-in” replication is initiated by redundant restart pathways (violet arrows) that are, respectively, PriA helicase- and PriB-dependent (subpanel 2), finally resulting in successful reconstitution of two intact chromosomes (subpanel 3). (B) In ΔIF2-1 mutant, steps until initiation of RecA-mediated strand invasion are similar to those above (subpanel 1). The oriC-to-Ter directed replisome is established through the redundant restart pathways, but that from Ter-to-oriC is not assembled (subpanel 2). Copy number at lacZ is restored by the former, but two-ended DSB repair fails to be consummated (subpanel 3).