Near-continuously synthesized leading strands in Escherichia coli are broken by ribonucleotide excision

Abstract

In vitro, purified replisomes drive model replication forks to synthesize continuous leading strands, even without ligase, supporting the semidiscontinuous model of DNA replication. However, nascent replication intermediates isolated from ligase-deficient Escherichia coli comprise only short (on average 1.2-kb) Okazaki fragments. It was long suspected that cells replicate their chromosomal DNA by the semidiscontinuous mode observed in vitro but that, in vivo, the nascent leading strand was artifactually fragmented postsynthesis by excision repair. Here, using high-resolution separation of pulse-labeled replication intermediates coupled with strand-specific hybridization, we show that excision-proficient E. coli generates leading-strand intermediates >10-fold longer than lagging-strand Okazaki fragments. Inactivation of DNA-repair activities, including ribonucleotide excision, further increased nascent leading-strand size to ∼80 kb, while lagging-strand Okazaki fragments remained unaffected. We conclude that in vivo, repriming occurs ∼70× less frequently on the leading versus lagging strands, and that DNA replication in E. coli is effectively semidiscontinuous.

Keywords: Okazaki fragments; ligase mutant; replication intermediates; ribonucleotide excision repair; the leading strand.

Fig. 1.

Continuously synthesized leading strand may appear discontinuous due to excision repair. (A) A scheme of fully discontinuous and semidiscontinuous replication highlighting the possible effects of excision repair. (B) Analysis by high-resolution alkaline-sucrose gradient of ligase-deficient replication intermediates reveals a substantial shoulder population of higher molecular weight intermediates. Here and henceforth, all gradients are sedimented from right (Top) to left (Bottom), with HMW species on the left, and LMW species on the right. The main panel shows the distribution of the [³H]dT pulse-labeled replication intermediates (red; at 42 °C for 2 min) or chronically labeled total chromosomal DNA (blue; at 28 °C for 35 min). (B, Bottom) The position of ³²P-labeled molecular weight markers of the individual gradients (typically MWM-9, which is a 9.3-kb PCR fragment), color matched to the main panel. Specifically, for this panel, the strain is lig (GR501). The values are means ± SEM of 31 to 44 independent repetitions. Due to the significant number of repetitions, most error bars are completely masked by the symbols. (C) Same as in B, but the strain is lig ZER (LA111, the excision-minus GR501). The values are means ± SEM of four to nine independent repetitions. (D) The rnhB mutants accumulate significant density of single rNs in their DNA, as measured by supercoiled plasmid relaxation with RNase HII in vitro. RD, relaxed dimer; RM, relaxed monomer; SCD, supercoiled dimer; SCM, supercoiled monomer. Strains: WT, AB1157; rnhB, L-415. (E) Accumulation of alkaline-sensitive material in the DNA of excision-minus, rnhB, or combined mutants. Unlike in D, plasmid DNA was linearized with MluI and then treated as follows: nt, no treatment; 0°, 0.2 M NaOH, 0 °C × 5 min (background #1); F, 100% formamide, 37 °C × 5 min (background #2); 45°, 0.3 M NaOH, 45 °C × 90 min. Plasmid species: dsl, double-strand linear; ssl, single-strand linear. Strains: lig, GR501; lig ZER, LA111; lig ∆B (GR501 ∆rnhB), eGC193; lig ZER ∆B, eGC197. (F) The density of alkaline-sensitive sites was determined from several gels such as in E and is presented as the number per genome equivalent (±SEM). (G) Neutral 0.8% agarose gel separation of the chromosomal DNA prepared in agarose plugs and denatured in 0.2 M NaOH at 0 °C for 30 min to reveal any ss breaks. An inverted ethidium bromide (EtBr)-stained image is shown. As a positive control for ss-break detection, we incubated the two control lanes, marked HL, in 0.3 M NaOH at 45 °C for 90 min to quantitatively hydrolyze single DNA-rNs; this nicks on average every 10 kb of the DNA strands of the lig ∆B mutant; the lig DNA remains intact. Strains are like in E.

Fig. 2.

Formamide-urea-sucrose gradients reveal HMW replication intermediates in the lig ZER ∆B mutant. (A) The separation pattern of FUS gradients for the lig mutant (GR501). The temperature of [³H]dT labeling is indicated. The corresponding alkaline-sucrose gradients from Fig. 1B are shown in faded colors for comparison. (B) The effect of removal of DNA-rN excision (lig ∆B) versus all other excision-repair systems (lig ZER) in FUS gradients. Strains: lig ZER, LA111; lig ∆B, eGC193. (C) The effect of the removal of both DNA-rN excision and all other excision-repair systems in one strain (lig ZER ∆B, eGC197) in FUS gradients. (D) FUS-gradient separation of our standard molecular weight markers: the 9.3-kb PCR fragment, phage lambda (48.5 kb), and phage T4 (∼170 kb), relative to the Okazaki fragments peak (∼1.1 kb) from the lig mutant (GR501).

Fig. 3.

Alkaline-sucrose gradients, sedimented at 4 °C, detect HMW replication intermediates. (A) The relative stability of RNA in alkaline solution at 0 °C. Incubation was for 15 min in the indicated conditions (either 0.2 M NaOH, preneutralized 0.2 M NaOH, or formamide-urea loading buffer), at the indicated temperatures (either 0, 16, or 37 °C). Control, input total E. coli RNA (mostly 23S+16S+5S rRNA). The reversed image of EtBr-stained 1.2% agarose gel is shown, to visualize the kilobase ladder. (B) The FUS-gradient separation of replication intermediates from the four ligase-deficient mutants with increasing levels of excision-repair deficiency. Strains: lig, GR501; lig ∆B, eGC193; lig ZER, LA111; lig ZER ∆B, eGC197. (C) When sedimented through chilled AS gradients, genomic DNA from the rnhB mutant displays a reduced size in comparison with WT, presumably due to limited alkaline hydrolysis of DNA-rNs. Vertical arrows indicating peak fractions are color matched to the corresponding gradients. Mean ± SEM for multiple experiments [lig (GR501) n = 44; lig ∆B (eGC193) n = 10]. (D) As in B, but AS gradients at 4 °C. Each curve represents the mean of between 7 and 45 separate experiments; error bars are omitted for clarity. The visible shift to the right of the HMW peaks compared with the FUS gradients in B is ostensibly due to DNA-rN hydrolysis.

Fig. 4.

Strand-specific hybridization of the HMW versus LMW replication intermediates. Strand-specific hybridizations were performed on tritiated DNA isolated directly from alkaline sucrose-gradient fractions. Portions of all gradient fractions were counted to generate the gradient profiles, while the remainder of selected fractions (colored rectangles) were pooled and hybridized against complementary strand-specific ssRNA targets. The specificity (lead/lag) ratios found in the hybridization experiments are shown as pie charts (Top), above the corresponding boxes indicating the pooled fractions used for hybridization. Results are shown as the mean of three independent experiments; error bars represent SEM (see Fig. 5C for hybridization SEMs; nonnormalized hybridization data are presented in SI Appendix, Fig. S9). (A) Excision repair-proficient strain lig (GR501) was labeled with tritiated thymidine either at 28 °C for 35 min (bulk genomic DNA, blue curve) or 42 °C for 2 min (replication intermediates, red curve). Fractions 5 to 13 from the 28 °C gradient were hybridized to generate the specificity ratio shown in the Left pie chart, while fractions 8 to 20 and 23 to 27 from the 42 °C labeling were hybridized to produce the Center and Right pie charts, respectively. (B) As in A, but the excision repair-deficient strain lig ZER ∆B (eGC197) was labeled at 42 °C for 2 min, and the resulting replication intermediates were pooled from gradient fractions 8 to 20 and 23 to 27 and hybridized to produce the strand-specificity pie charts (shown, correspondingly, Center and Right).

Fig. 5.

HMW or LMW character of the RIs in Watson or Crick strands depends on the direction of DNA replication through the hybridization region. (A) The relative position of the gsp locus versus oriC on the chromosome (Top Left) ensures that the lagging nascent strand (LMW) is Watson, while the leading nascent strand (HMW) is Crick. The question is: If we switch the direction of the replication fork through the gsp region by using oriF (Top Right), will it make the nascent Watson strand HMW while the nascent Crick strand LMW? (B) Strand-specific hybridization of the HMW versus LMW replication intermediates in the oriF-driven strain. The strain PO45 is ligA251(Ts) ∆rnhB oriF+ ∆oriC (eGC225). (B, Left) The pie chart shows the relative leading-strand (blue) and lagging-strand (cyan) components of bulk chromosomal DNA isolated from the oriF-driven strain labeled at 28 °C (Lig+ conditions) (fractions 5 to 13 of the blue gradient). The other two pie charts display the strand bias for RIs isolated from fractions 8 to 20 (Middle) and 23 to 27 (Right), from the same oriF-driven strain labeled at 42 °C. (C) Summary hybridization data for Watson and Crick strand specificities of bulk chromosomal DNA (28 °C; gray bars) and RIs (42 °C), as assayed at the gsp locus, for the lig ZER ∆B mutant (eGC197) replicated from oriC (back row, light blue) versus PO45 (eGC225) replicated from oriF (front row, light purple). The oriC-driven strain hybridization data are from Fig. 4B, while the oriF-driven strain data are from B. Data are the mean ± SEM, n = 3 for all cases.

Fig. 6.

Evidence for periodic priming on the leading strand. (A) If the leading strands were replicated continuously, with no regular repriming events (Left), incorporation of [³H]thymidine into the nascent leading strands is expected to produce tritiated HMW species whose length is independent of the labeling pulse time (since nascent DNA is always attached to the rest of the chromosome). On the other hand, if the leading strands were replicated in smaller subchromosome-sized fragments (Right), shorter pulses would reduce the apparent size of nascent leading-strand fragments. (B) AS gradient of the lig ZER ∆B mutant (eGC197), either labeled chronically (28 °C × 35 min) for full-length chromosomal DNA or pulse labeled at 45 °C for 10, 30, 90, or 270 s to label RIs.