Rep provides a second motor at the replisome to promote duplication of protein-bound DNA

Abstract

Nucleoprotein complexes present challenges to genome stability by acting as potent blocks to replication. One attractive model of how such conflicts are resolved is direct targeting of blocked forks by helicases with the ability to displace the blocking protein-DNA complex. We show that Rep and UvrD each promote movement of E. coli replisomes blocked by nucleoprotein complexes in vitro, that such an activity is required to clear protein blocks (primarily transcription complexes) in vivo, and that a polarity of translocation opposite that of the replicative helicase is critical for this activity. However, these two helicases are not equivalent. Rep but not UvrD interacts physically and functionally with the replicative helicase. In contrast, UvrD likely provides a general means of protein-DNA complex turnover during replication, repair, and recombination. Rep and UvrD therefore provide two contrasting solutions as to how organisms may promote replication of protein-bound DNA.

Figure 1

Rep and UvrD Promote Replication Fork Movement through EcoRI E111G-DNA Complexes In Vitro (A) Relative positions of oriC and EcoRI sites within plasmid templates, the site of cleavage by EagI, and the predicted sizes of leading strand products generated with and without replication blockage. (B) Denaturing agarose gel of replication products from pPM594 with and without E111G in the presence of the indicated helicases/translocases. (C) Levels of the 4.7 kb leading strand generated from pPM594 in the presence of E111G and the indicated enzymes relative to control reactions in lanes 1 and 2 in (B). (D) Replication products with pME101. (E) Levels of the 4.7 kb leading strand generated with pME101, plus E111G and the indicated enzymes relative to control reactions in lanes 1 and 2 in (D). (F) Relative levels of the 4.7 kb leading strand generated with pME101 plus E111G at increasing concentrations of Rep and UvrD. E111G was present at 200 nM dimers in all assays, while helicases/translocases were at 100 nM unless indicated otherwise. Error bars represent standard deviation of the mean.

Figure 2

Analysis of Δrep ΔuvrD Lethality (A) Retention or loss of pAM407 (pRC7uvrD) in strains bearing mutations in rep and/or uvrD as judged by blue/white colony color on LB (top row) and minimal agar (bottom row) containing X-gal and IPTG. Fractions of white colonies are indicated below each image with the actual number of white colonies and total colonies shown in parentheses. (B) Retention or loss of pAM407 from Δrep ΔuvrD strains bearing a suppressor mutation identified subsequently as rpoB[T3713C], encoding RNAP L1238P (a); recF (b); recQ (c); recJ (d); and recA (e). Note that the suppressor strain N7124 in subpanel (a) was N7122, isolated as described in the text, into which pAM407 was reintroduced. (C) Strategy for the analysis of rich medium viability of plasmidless strains isolated on minimal agar. (D) The plasmid-free strains indicated were grown in liquid minimal medium before dilution and spotting onto LB or minimal agar.

Figure 3

Suppression of Δrep ΔuvrD Lethality by Mutations that Destabilize Transcription Complexes (A–D) Retention or loss of pAM407 in Δrep ΔuvrD strains bearing mutations in rpoB (A, B, and C) or spoT (D) on LB/X-gal/IPTG. Note that rpoB[C2489T] was isolated in this study, as described in the text, as N7181. N7208 was formed by reintroduction of pAM407 into N7181.

Figure 4

Suppressed Δrep ΔuvrD Cells Remain Hypersensitive to an Artificial Nucleoprotein Barrier to Replication (A–D) Strains PM462–465 bearing 34 chromosomal lac operators plus pPM306, a plasmid bearing lacI under the control of an arabinose-inducible promoter, were tested for colony-forming ability upon expression of lac repressor. Cells were grown in LB in the absence of arabinose, and then serial dilutions were spotted onto LB agar containing arabinose without and with IPTG.

Figure 5

Correlation between Helicase-Mediated Promotion of Fork Movement In Vitro and Complementation of Δrep ΔuvrD Lethality In Vivo (A) Denaturing agarose gel of replication products from pME101 (two EcoRI sites) with and without E111G (200 nM dimers) in the presence of the indicated helicases (100 nM). (B) Levels of the 4.7 kb leading strand in the presence of the indicated helicases relative to control reactions in lanes 2 and 3 in (A). Error bars represent standard deviation of the mean. (C) Scheme for generation of strains containing helicase genes under the control of an arabinose-inducible promoter. (D and E) Colony-forming ability of rep⁺uvrD⁺ (N6524) and Δrep ΔuvrD (N6556) strains lacking pRC7rep but bearing the indicated plasmids after growth in liquid minimal medium, subsequent dilution, and spotting onto LB containing kanamycin ± arabinose.

Figure 6

Rep Interacts with DnaB (A) Binding of Rep and UvrD to surface-immobilized E. coli and B. stearothermophilus DnaB (860 and 1705 resonance units, respectively), as measured by surface plasmon resonance. Concentrations of Rep and UvrD were 200, 500, 1000, 2000, and 4000 nM. (B) Coomassie-stained gel of pull-down assays from whole-cell extracts with biotinylated Rep, RepΔC33, and UvrD as bait (subpanel [a], lanes 5–7, respectively). Lanes 2 and 3 contained 100 ng of purified DnaB and 12 μg of untreated whole-cell extract, respectively, while protein from a mock pull-down experiment with no bait protein present was run in lane 4 (a). Subpanel (b) shows a western blot, using DnaB antibodies, of the pull-down assays shown in (a). (C) Binding of Rep and E. coli DnaB to forked DNA having two ssDNA arms. Concentrations of Rep were 1, 5, 10, and 25 nM, while DnaB was present at 100 nM hexamers as indicated. (D) Binding of UvrD and DnaB to forked DNA. Concentrations of UvrD were as for Rep in (C), and the concentration of DnaB was 100 nM hexamers. (E) Unwinding of forked DNA by E. coli DnaB (100 nM hexamers), Rep, and UvrD (both at 10 nM). (F) Fraction of the forked DNA substrate unwound by the indicated helicases. Protein concentrations were as in (E). Error bars represent standard deviation of the mean. (G) Relative levels of forked DNA unwinding by DnaB plus Rep/UvrD (“HelX”) in comparison to the sum of unwinding by each individual helicase. Protein concentrations were as in (E). (H) Relative levels of forked DNA unwinding by 100 nM DnaB hexamers in the presence of the indicated concentrations of Rep and UvrD. Concentrations >10 nM were not tested due to high levels of unwinding by UvrD. (I) Inhibition of the DnaB-Rep interaction by DnaC; binding of DnaB (1 μM monomers) and/or DnaC (4 μM monomers) to the C33^Rep peptide (260 resonance units surface-immobilized via an N-terminal biotin tag). (J) Interaction of DnaB with C33^Rep in the presence of increasing concentrations of DnaC. Conditions were otherwise as in (I).

Figure 7

Promotion of Rep Function In Vitro and In Vivo by Interaction with DnaB (A) Denaturing agarose gel of replication products from pPM594 (eight EcoRI sites) with and without E111G (200 nM dimers) in the presence of Rep and RepΔC33 (100 nM). Note that RepΔC33 also failed to promote replisome movement along template bearing two EcoRI sites (data not shown). (B) Levels of the 4.7 kb leading strand in the presence of the indicated helicases relative to control reactions in lanes 1 and 2 in (A). Error bars represent standard deviation of the mean. (C and D) Colony-forming ability of rep⁺ uvrD⁺ (N6524) and Δrep ΔuvrD (N6556) strains bearing the indicated plasmids, tested as described in Figure 5C.