RdgB acts to avoid chromosome fragmentation in Escherichia coli

Abstract

SummaryBacterial RecA protein is required for repair of twostrand DNA lesions that disable whole chromosomes. recA mutants are viable, suggesting a considerable cellular capacity to avoid these chromosomedisabling lesions. recA -dependent mutants reveal chromosomal lesion avoidance pathways. Here we characterize one such mutant, rdgB / yggV , deficient in a putative inosine/xanthosine triphosphatase, conserved throughout kingdoms of life. The rdgB recA lethality is suppressed by inactivation of endonuclease V (gp nfi ) specific for DNA-hypoxanthines/ xanthines, suggesting that RdgB either intercepts improper DNA precursors dITP/dXTP or works downstream of EndoV in excision repair of incorporated hypoxathines/xanthines. We find that DNA isolated from rdgB mutants contains EndoV-recognizable modifications, whereas DNA from nfi mutants does not, substantiating the dITP/dXTP interception by RdgB. rdgB recBC cells are inviable, whereas rdgB recF cells are healthy, suggesting that chromosomes in rdgB mutants suffer double-strand breaks. Chromosomal fragmentation is indeed observed in rdgB recBC mutants and is suppressed in rdgB recBC nfi mutants. Thus, one way to avoid chromosomal lesions is to prevent hypoxanthine/xanthine incorporation into DNA via interception of dITP/dXTP.

RecA-dependent mutants in Escherichia coli reveal strategies to avoid chromosomal fragmentation

“…To test our prediction that all RecA-dependent mutants suffer from increased chromosomal fragmentation, we introduced the isolated mutations into recBC(Ts) strain SK129, defective at 37°C in both the RecBCD-promoted linear DNA degradation and RecBC-catalyzed recombinational repair of double-strand breaks (30). The isolated RecA-dependent mutants proved to be inhibited at 42°C when introduced into the recBC(Ts) strain (data not shown), indicating their dependence on the RecBCD enzyme, as is generally the case with known RecA-dependent mutants (20,24,31). We measured the degree of chromosomal fragmentation in the double mutants using pulsed-field gel electrophoresis (Fig.…”

“…Growing cultures (OD 600 for 37°C samples varied from 0.7 to 1.1; for 22°C samples, variation was from 0.4 to 0.6) were normalized to OD 600 0.35, and 0.5 ml of the normalized cultures was pelleted and washed once with 1 ml of TE buffer (10 mM Tris͞1 mM EDTA, pH 8.0) to remove unincorporated radioactivity. Plug preparation was as described (20). Half of the plug was inserted in 1% agarose gel on 0.5 ϫ TBE buffer (89 mM Tris͞89 mM boric acid͞2.0 mM EDTA, pH 7.8) and run for 24 h at 6 V͞cm with a switch time ramp 60-120 seconds, in CHEF-DRII Pulsed-Field Gel Electrophoresis (BioRad).…”

“…Replication-caused transient hemimethylation of GATC sites is used by E. coli to identify the old DNA strand as the proper template for mismatch repair, whereas lack of GATC methylation in dam mutants results in double-strand DNA breaks due to a disoriented mismatch repair (18). The rdgB mutants depend on RecA and degrade their chromosomal DNA in recA Ϫ conditions (19), because they are deficient in ITP͞XTPase, an enzyme believed to intercept the improper DNA precursor dITP, thus preventing hypoxanthine incorporation into DNA (20). In the absence of RdgB, hypoxanthine is proposed to occasionally incorporate into DNA in place of guanine, its subsequent excision by EndoV translating into chromosomal fragmentation (20).…”

“…The rdgB mutants depend on RecA and degrade their chromosomal DNA in recA Ϫ conditions (19), because they are deficient in ITP͞XTPase, an enzyme believed to intercept the improper DNA precursor dITP, thus preventing hypoxanthine incorporation into DNA (20). In the absence of RdgB, hypoxanthine is proposed to occasionally incorporate into DNA in place of guanine, its subsequent excision by EndoV translating into chromosomal fragmentation (20). Last, fur mutants are deficient in the repression of iron assimilation and, as a result, experience iron overload (21), which is postulated to lead to increased oxidative DNA damage and RecA dependence in aerobic conditions (22).…”

Molybdenum cofactor-dependent resistance to N-hydroxylated base analogs in Escherichia coli is independent of MobA function

“…Ham1p is a specific pyrophosphatase that hydrolyzes HAP deoxyribonucleoside triphosphate to the corresponding mononucleotide, thus preventing incorporation of the analog into DNA [4,8]. While E. coli contains a homolog for the HAM1 gene, named rdgB [9,10], this system apparently plays a back-up role in HAP detoxification [11,12]. Its main function is likely the removal of (d)ITP or (d)XTP from the cellular (d)NTP pools [10][11][12].…”