Functions that protect Escherichia coli from DNA–protein crosslinks

Abstract

Pathways for tolerating and repairing DNA-protein crosslinks (DPCs) are poorly defined. We used transposon mutagenesis and candidate gene approaches to identify DPC-hypersensitive Escherichia coli mutants. DPCs were induced by azacytidine (aza-C) treatment in cells overexpressing cytosine methyltransferase; hypersensitivity was verified to depend on methyltransferase expression. We isolated hypersensitive mutants that were uncovered in previous studies (recA, recBC, recG, and uvrD), hypersensitive mutants that apparently activate phage Mu Gam expression, and novel hypersensitive mutants in genes involved in DNA metabolism, cell division, and tRNA modification (dinG, ftsK, xerD, dnaJ, hflC, miaA, mnmE, mnmG, and ssrA). Inactivation of SbcCD, which can cleave DNA at protein-DNA complexes, did not cause hypersensitivity. We previously showed that tmRNA pathway defects cause aza-C hypersensitivity, implying that DPCs block coupled transcription/translation complexes. Here, we show that mutants in tRNA modification functions miaA, mnmE and mnmG cause defects in aza-C-induced tmRNA tagging, explaining their hypersensitivity. In order for tmRNA to access a stalled ribosome, the mRNA must be cleaved or released from RNA polymerase. Mutational inactivation of functions involved in mRNA processing and RNA polymerase elongation/release (RNase II, RNaseD, RNase PH, RNase LS, Rep, HepA, GreA, GreB) did not cause aza-C hypersensitivity; the mechanism of tmRNA access remains unclear.

Stimulation of Replication Template-Switching by DNA-Protein Crosslinks

“…RecA-independent and does not require DSB repair: The ability to repair double-strand DNA breaks through the RecA RecBCD-dependent homologous recombination pathway has been shown to promote survival to 5-azaC [30][31][32][33][34]. We confirmed the recA and recB were required to sustain viability during a 2 hour treatment with 5-azaC at various concentrations (Fig.…”

“…DPCs can lead to the formation of double-strand breaks in mammalian cells [47]. In bacteria, tolerance of DPCs is diminished in recA and recBCD mutants [30][31][32][33][34], strains defective in DSB repair. RecA is not, however, required for the recovery of template-switch mutations induced by Figure 11.…”

Functions that Protect Escherichia coli from Tightly Bound DNA-Protein Complexes Created by Mutant EcoRII Methyltransferase

“…Recombination and repair proteins play important roles in survival from damage due to DPCs and topoisomerase cleavage complexes. For example, RecA, which catalyzes strand exchange, and RecBCD, which prepares broken ends for HR, greatly improve survival from aza-C-induced DPCs [ 8 , 46 , 47 , 49 , 52 , 65 ] and quinolone-induced cleavage complexes [ 66 , 67 ]. We found that recA , recB and recC mutants were all markedly sensitive to TBCs generated by expression of M.EcoRII-C186A ( Fig 3 ; data summarized in Table 1 ).…”

“…E . coli transposon mutants (kanamycin resistance gene) were created in a recent genetic screen [ 51 , 52 ] and are identified here as insertions (e.g. ssrA :: Kan ).…”

“…We also performed an extensive candidate gene screen for mutants that are hypersensitive to the M.EcoRII-induced solo TBCs, using an arabinose expression system that allows carefully titrated expression of M.EcoRII. A powerful aspect of this system is that previous studies, from our lab and others, have already identified a variety of mutants that are hypersensitive to aza-C-induced DPCs that involve the same M.EcoRII protein (except that the DPCs utilize the wild-type version of the protein) ([ 8 , 46 , 47 , 48 , 49 , 50 , 51 , 52 ]; reviewed in [ 7 ]). We also took the opportunity to test the sensitivity of each mutant to two of the quinolone antibiotics, which target the bacterial type II topoisomerases [ 53 , 54 , 55 ].…”