UvrD helicase, unlike Rep helicase, dismantles RecA nucleoprotein filaments in Escherichia coli

Abstract

The roles of UvrD and Rep DNA helicases of Escherichia coli are not yet fully understood. In particular, the reason for rep uvrD double mutant lethality remains obscure. We reported earlier that mutations in recF, recO or recR genes suppress the lethality of uvrD rep, and proposed that an essential activity common to UvrD and Rep is either to participate in the removal of toxic recombination intermediates or to favour the proper progression of replication. Here, we show that UvrD, but not Rep, directly prevents homologous recombination in vivo. In addition to RecFOR, we provide evidence that RecA contributes to toxicity in the rep uvrD mutant. In vitro, UvrD dismantles the RecA nucleoprotein filament, while Rep has only a marginal activity. We conclude that UvrD and Rep do not share a common activity that is essential in vivo: while Rep appears to act at the replication stage, UvrD plays a role of RecA nucleoprotein filament remover. This activity of UvrD is similar to that of the yeast Srs2 helicase.

Figure 1

(A) Scheme of the replication assay. ssDNA is passing from the donor cell (left side) to the recipient cell (right side), in which it is converted to dsDNA (dotted arrow lines, oriented 5′–3′). In the donor strain (left side), the LacI^s protein (small grey dots) represses strongly the lacZ promoter, so that almost no LacZ is produced. In the recipient strain, as soon as the incoming DNA is replicated, a burst of LacZ synthesis occurs (big grey circle), due to the initial absence of LacI^s. After a 1 h delay, sufficient amounts of LacI^s are produced to shut down lacZ transcription. (B) Scheme of the recombination assay. Recombination occurs between the incoming lacZΔCt allele and the lacZΔNt allele of the recipient chromosome (black boxes), and leads to a functional gene and enzyme production (big grey circle).

Figure 2

Kinetics of recombination (A, C, E) and replication (B, D, F) after conjugation in various mutants. Replication and recombination were monitored by using donor strains Nec226 for the replication assay and Nec224 for the recombination assay, as described in the text. All experiments were repeated at least three times. The recipient strains are designated by an alias drawn near each curve: ‘wt’ Nec223 (○), ‘recA’ Nec229 (◊), ‘recB’ Nec228 (∇), ‘recF’ Nec227 (▴), ‘recB recF’ Nec232 (✗), ‘recA recD’ Nec230 (), ‘uvrD’ MAC1058 (□), ‘pBR’ MAC1068 (Δ), ‘UvrD++’ MAC1071 (⧫) and ‘rep’ MAC1065 (▪). The ‘control’ curves in panels A, C and E (•) represent the cross between two identical lacZΔP alleles, Nec223 and Nec225 strains, which cannot result in a Lac+ recombinant. The ‘control’ curve in panels B, D and E represents the Hfr3000 lacI^s donor strain without any recipient strain (•): it indicates the amount of transcription leakage in the presence of IPTG, with the lacI^s allele (around 5 units). After a 40 min conjugation period on filter, β-galactosidase activity of cell extracts was measured as a function of incubation time at 28°C.

Figure 3

The sfiA rep uvrD recF recA730 (MAC1159) grows poorly compared to its recA+ parent (MAC1168). Both strains were streaked on an LB plate and incubated for 48 h at 37°C.

Figure 4

UvrD helicase but not Rep helicase inhibits strand exchange catalysed by RecA. (A) Scheme of DNA strand exchange reaction (ssc, single-stranded circular DNA; dsl, double-stranded linear DNA; jm, joint molecules; nc, nicked circular double-stranded DNA; ssl, single-stranded linear DNA). (B) After RecA nucleoprotein filament formation by preincubation of ssDNA with RecA and SSB proteins, various amounts of UvrD helicase were added simultaneously with ³²P end-labelled linear dsDNA, which initiates the strand exchange (lane 1: labelled dsl; lanes 2–7 correspond to 0, 600, 300, 150, 75, 37.5 and 18.75 nM UvrD, respectively). After incubation for 40 min at 37°C, reaction mixture was deproteinized and resolved onto a 0.8% agarose gel. (C) Same as in (B) except that Rep was used in place of UvrD (lanes 1–6 correspond to 0, 1400, 1000, 600, 300 and 150 nM Rep, respectively). (D) Quantification of the reactions shown in panels B and C. (E) Comparable ATPase activities of UvrD and Rep proteins. Reactions containing 1 mM ATP, saturating amount of synthetic oligo(dT)₅₅ (5.5 μM nucleotides) and increasing amount of proteins were incubated at 37°C for 15 min. The calculated amount of ATP hydrolysed linked to the oxidation of NADH was determined by measuring the OD at 340 nm.

Figure 5

UvrD helicase, but not Rep helicase, efficiently disrupts RecA–ssDNA nucleoprotein filament. (A) RecA–ssDNA nucleoprotein filaments. (B) Preformed RecA–ssDNA complexes were incubated for 15 min with UvrD. The arrows point to the ssDNA covered with SSB. (C) Blow-up of ssDNA covered with SSB. (D) The percentage of disrupted RecA presynaptic filament was determined for different amounts of both helicases. For each concentration, mean values and standard deviations were obtained from two series of 300 molecules counted. Scale bars, 50 nm.