Exponential propagation of large circular DNA by reconstitution of a chromosome-replication cycle

Abstract

Propagation of genetic information is a fundamental property of living organisms. Escherichia coli has a 4.6 Mb circular chromosome with a replication origin, oriC. While the oriC replication has been reconstituted in vitro more than 30 years ago, continuous repetition of the replication cycle has not yet been achieved. Here, we reconstituted the entire replication cycle with 14 purified enzymes (25 polypeptides) that catalyze initiation at oriC, bidirectional fork progression, Okazaki-fragment maturation and decatenation of the replicated circular products. Because decatenation provides covalently closed supercoiled monomers that are competent for the next round of replication initiation, the replication cycle repeats autonomously and continuously in an isothermal condition. This replication-cycle reaction (RCR) propagates ∼10 kb circular DNA exponentially as intact covalently closed molecules, even from a single DNA molecule, with a doubling time of ∼8 min and extremely high fidelity. Very large DNA up to 0.2 Mb is successfully propagated within 3 h. We further demonstrate a cell-free cloning in which RCR selectively propagates circular molecules constructed by a multi-fragment assembly reaction. Our results define the minimum element necessary for the repetition of the chromosome-replication cycle, and also provide a powerful in vitro tool to generate large circular DNA molecules without relying on conventional biological cloning.

Figure 1.

Schematic representation of the RCR reconstituted with purified proteins.

Figure 2.

Replication reaction of oriC-circular DNA in the presence of decatenation enzymes. (A) oriC-containing supercoiled DNA (M13ms10, 150 pM as supercoiled circular DNA, 24 pmol as nucleotides in 10 μl reaction) was incubated in RCR mix (containing [α-³²P]dATP) at 30°C for 1 h in the presence or absence of either Topo IV or Topo III–RecQ. DNA products were analyzed by agarose-gel electrophoresis. (B) The amount of total DNA synthesis. dNTP incorporation was quantified and values were normalized to the amount of input supercoiled template as nucleotide (fold input, closed circles). The ratio of supercoiled:total products was determined from the band intensities in the gel image, enabling calculation of the amount of supercoiled product normalized to input supercoiled template from the amount of total DNA synthesis (fold input, open circles). (C) RecQ stimulates Topo III decatenation activity in RCR. oriC-containing circular DNA (M13ms10, 150 pM) was incubated in the RCR mixture, excluding Topo IV, at 30°C for 20 min in the presence of the indicated concentrations of Topo III and/or RecQ. [α-³²P]dATP-incorporated DNA was then detected. The compositions of the RCR mixture are listed in Supplementary Table S4.

Figure 3.

Continuous repetition of the RCR. (A) The indicated amount of pOri8 (9.5 kb) was incubated in the RCR mixture for 3 h. Aliquots (1 μl) were detected by agarose-gel electrophoresis and SYBR Green staining. The input DNA (10⁹ molecules in 10 μl, 1 ng/μl) was also detected (‘no RCR’). Size-marker fragments (M1) were derived from phage λ DNA. (B) pOri8 (10⁵ molecules/μl) was incubated in the RCR mixture at 30°C. Aliquots (1 μl) were taken at the indicated times and the number of DNA circles was quantified using the transformation method. The RCR mixture was preincubated at 30°C for 15 min before the addition of template DNA. The values from three independent reactions are shown with the error bars (standard error of the mean). (C) pPKOZ (10⁻⁵ ng, 8.9 kb) was incubated in the RCR mixture for 3 h (passage 1). The sample was then diluted 10⁶-fold in the fresh RCR mixture and further incubated for 3 h (passage 2). This sequence was repeated for a total of 10 incubations. At each passage, aliquots were analyzed. Size-marker fragments (M1) were derived from phage λ DNA. DNA was visualized by staining with SYBR Green I. (D) The total doublings were deduced using the transformation method. Error rates per base per replication cycle were deduced by blue–white determination of the lacZ status (28). Mutagenic dNTPs, when included, consisted of 1 μM each of 8-oxo-GTP and dPTP. The transformation analysis and the blue–white determination were performed twice and the mean values were shown with the ranges.

Figure 4.

Monoclonal propagation of DNA by limiting-dilution. Mixtures of pOri8 (9.5 kb) and pOriDif (12 kb) after limiting dilution (15 molecules of each in samples 1–3, an average of 1.5 molecules of each in samples 4–7) in the RCR mixture (10 μl) were incubated for 6 h. pOri8 and pOriDif were also individually propagated. Size-marker fragments (M1) were derived from phage λ DNA. DNA was visualized by staining with SYBR Green I.

Figure 5.

Propagation of large, circular DNA in RCR. (A) ‘Pop-out’ method for generating large, oriC-containing circular DNAs. An 80 or 200 kb chromosomal region containing oriC was retrieved via the parABC-Km cassette in cells expressing the λ Red genes. The structure of the isolated 205 kb DNA was verified by restriction enzyme analysis (Supplementary Figure S8). (B) A total of 85 kb (pOri80, 15 pM) or 205 kb (pOri200, 5 pM) oriC-containing circular DNA was incubated in the RCR mixture at 30°C for 3 h. Samples before (−) or after (+) RCR were analyzed by agarose-gel electrophoresis and SYBR Green staining. Size-marker fragments (M2) were derived from T7 DNA.

Figure 6.

Two-step cell-free cloning using RCR. Each of three PCR fragments (1 kb oriC, 3.3 kb lacZ and 4.6 kb parABC-Km; 1.5 fmol) containing 60 bp overlaps at both ends were incubated in modified Gibson Assembly mixture (5 μl) at 50°C for 1 h. Aliquots (1 μl) were then incubated in the RCR mixture (10 μl) at 30°C for 3 h. Samples without assembly and/or RCR were analyzed as negative controls (−). pPKOZ was propagated by RCR (‘Control DNA’). The assembly samples before or after RCR were also analyzed by E. coli transformation and blue-white determination. Size-marker fragments (M3) were derived from phage λ DNA.