Role for radA/sms in recombination intermediate processing in Escherichia coli - PubMed (original) (raw)

Role for radA/sms in recombination intermediate processing in Escherichia coli

Cynthia E Beam et al. J Bacteriol. 2002 Dec.

Abstract

RadA/Sms is a highly conserved eubacterial protein that shares sequence similarity with both RecA strand transferase and Lon protease. We examined mutations in the radA/sms gene of Escherichia coli for effects on conjugational recombination and sensitivity to DNA-damaging agents, including UV irradiation, methyl methanesulfonate (MMS), mitomycin C, phleomycin, hydrogen peroxide, and hydroxyurea (HU). Null mutants of radA were modestly sensitive to the DNA-methylating agent MMS and to the DNA strand breakage agent phleomycin, with conjugational recombination decreased two- to threefold. We combined a radA mutation with other mutations in recombination genes, including recA, recB, recG, recJ, recQ, ruvA, and ruvC. A radA mutation was strongly synergistic with the recG Holliday junction helicase mutation, producing profound sensitivity to all DNA-damaging agents tested. Lesser synergy was noted between a mutation in radA and recJ, recQ, ruvA, ruvC, and recA for sensitivity to various genotoxins. For survival after peroxide and HU exposure, a radA mutation surprisingly suppressed the sensitivity of recA and recB mutants, suggesting that RadA may convert some forms of damage into lethal intermediates in the absence of these functions. Loss of radA enhanced the conjugational recombination deficiency conferred by mutations in Holliday junction-processing function genes, recG, ruvA, and ruvC. A radA recG ruv triple mutant had severe recombinational defects, to the low level exhibited by recA mutants. These results establish a role for RadA/Sms in recombination and recombinational repair, most likely involving the stabilization or processing of branched DNA molecules or blocked replication forks because of its genetic redundancy with RecG and RuvABC.

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Figures

FIG. 1.

FIG. 1.

Synergy of radA with mutations affecting recombinational UV repair. Shown are UV survival curves for AB1157-derived strains both singly and doubly deficient for radA, recA, recJ, recQ, and recB. (A) AB1157, rec+ (▪); STL5280, radA1::kan (•); JC10287, recA_Δ (⧫); STL4799, recA_Δ radA1::kan (◊); (B) AB1157, rec+ (▪); STL5280, radA1::kan (•); STL1548, recQ1802::Tn_3 (▴); JC12123, recJ284::Tn_10 (⧫); STL5048, recQ1802::Tn_3 radA1_::kan (Δ); STL5042, recJ284::Tn_10 radA1_::kan (◊); (C) AB1157, rec+ (▪); STL5280, radA1::kan (•); N2101, recB268::Tn_10_ (▴); STL5480, recB268::Tn_10 radA1_::kan (Δ). Error bars indicate standard errors of the determinations.

FIG. 2.

FIG. 2.

RadA is synergistic with Holliday junction-processing genes in UV repair. UV survival curves are shown for E. coli strains both singly and multiply deficient for radA, recG, ruvA, and ruvC. All strains assayed for UV survival were derived from the AB1157 background. (A) AB1157, rec+ (▪); STL5280, radA1::kan (•); N2096, ruvAΔ63 (▴); CS140, ruvC53 (⧫); STL5046, ruvC53 radA1::kan (◊); STL5037, ruvAΔ63 radA1::kan (Δ); (B) AB1157, rec+ (▪); STL5280, radA1::kan (•); N4452, recGΔ265::cat (▴); STL6588, recGΔ265::cat radA1::kan (Δ); (C) AB1157, rec+ (▪); STL5280, radA1::kan (•); STL6586, recGΔ265::cat ruvC53 (◊); STL6640, recGΔ265::cat radA1::kan ruvC53 (⧫); STL6571, recGΔ265::cat ruvAΔ63(Δ); STL6592, recGΔ265::cat radA1::kan ruvAΔ63 (▴). Error bars indicate standard errors of the determinations.

FIG. 3.

FIG. 3.

Double-strand-break (DSB)-mediated recombination. A broken fork can be repaired by recombinational reactions. (Likewise, ends of conjugative DNA or transducing fragments can be integrated via this mechanism.) Double-strand ends are resected by RecBCD nuclease; RecBCD also assists in loading of RecA onto single-strand DNA. The RecA-single-strand DNA filament promotes strand invasion into a homologous duplex molecule (the sister chromosome), forming a D-loop intermediate. Branch migration helicases can extend the region of pairing to form a Holliday junction (HJ), which can be resolved by cleavage mediated by Holliday junction endonucleases such as RuvC. Ligation of strand scissions restores an intact recombinant chromosome. RadA may participate in recombination by stabilizing any of these joint intermediates or by mediating branch migration or cleavage.

FIG. 4.

FIG. 4.

Fork regression and repair. Lesions can stall DNA polymerase. Fork regression catalyzed by RecG or RuvAB helicase activity can move the lesion into double-strand DNA, where it can be repaired. 5′ to 3′ degradation of the lagging strand by RecJ/RecQ may facilitate repair when the lagging strand has moved ahead of the leading strand. The regressed fork forms a Holliday junction, which can be cleaved by RuvC. Double-strand break repair can then restore integrity of the fork. Alternatively, RecBCD degradation of the double-strand arm or reversed branch migration can restore a fork structure.

FIG. 5.

FIG. 5.

Alignment of E. coli RadA/Sms with human Dmc1 showing similarity through the putative Zn finger region. Arrows indicate the conserved cysteines of the Zn finger.

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