Efficient Arrangement of the Replication Fork Trap for In Vitro Propagation of Monomeric Circular DNA in the Chromosome-Replication Cycle Reaction

Abstract

Propagation of genetic information is a fundamental prerequisite for living cells. We recently developed the replication cycle reaction (RCR), an in vitro reaction for circular DNA propagation, by reconstitution of the replication cycle of the Escherichia coli chromosome. In RCR, two replication forks proceed bidirectionally from the replication origin, oriC, and meet at a region opposite oriC, yielding two copies of circular DNA. Although RCR essentially propagates supercoiled monomers, concatemer byproducts are also produced due to inefficient termination of the replication fork progression. Here, we examined the effect of the Tus-ter replication fork trap in RCR. Unexpectedly, when the fork traps were placed opposite oriC, mimicking their arrangement on the chromosome, the propagation of circular DNA was inhibited. On the other hand, fork traps flanking oriC allowed efficient propagation of circular DNA and repressed concatemer production. These findings suggest that collision of the two convergence forks through the fork trap is detrimental to repetition of the replication cycle. We further demonstrate that this detrimental effect was rescued by the UvrD helicase. These results provide insights into the way in which circular DNA monomers replicate repetitively without generating concatemers.

Keywords: DNA amplification; chromosome replication; replication fork; replication termination.

Figure 1

Effect of Tus on the RCR propagation of circular DNA in which the oriC-ter arrangement mimics the chromosome position. pKZter_1 (+ter) is a pKOZ (−ter) derivative containing inward-facing ter sites opposite oriC. The regions available for fork progression from the oriC cassette are indicated by dotted arrows on the circular map. pPKOZ or pKZter_1 (2.5 ng) was incubated in the RCR mixture at 30 °C for 3 h in the presence of the indicated concentrations of Tus. Aliquots (0.2 µL) were analyzed by 0.5% TBE-agarose gel electrophoresis and SYBR Green I staining. The linear DNA size marker fragments were derived from lambda phage DNA (marker).

Figure 2

Effect of the inter-ter length on the RCR propagation in the presence of Tus. (A) pKZter_2–6 are pKZter_1 derivatives in which the length between the inward-facing ter sites was extended to 0.5, 1.0, 2.0, 4.0, or 10 kb. The circular maps are shown as in Figure 1. The indicated plasmid (2.5 ng) was incubated in the RCR mixture at 30 °C for 3 h in the absence (0 nM) or presence (20 nM) of Tus. The product was analyzed by 0.5% TBE-agarose gel electrophoresis and SYBR Green I staining. The ratio of concatemers to the sum of concatemers and supercoils is shown in a graph as the average value of three repetitive experiments with standard deviation. (B) In pCLter30k, the regions available for fork progression were 15 kb (dotted arrows), while the inter-ter length was 0.2 kb. RCR was performed as in (A), except that a 1% agarose gel was used to separate large supercoiled DNAs from concatemers. Large supercoiled DNA migrates more slowly than linear DNA [29]. DNA size marker fragments were derived from lambda phage DNA (marker).

Figure 3

RCR propagation of circular DNA containing ter sites on both sides of oriC. The circular maps of pCLter_1 and pCLter_2 are shown as in Figure 1. The indicated plasmid (2.5 ng) was incubated in the RCR mixture at 30 °C for 3 h in the absence (0 nM) or presence (6, 20, or 60 nM) of Tus. The product was analyzed by 0.5% TBE-agarose gel electrophoresis and SYBR Green I staining. The ratio of concatemers to the sum of concatemers and supercoils is shown in a graph.

Figure 4

Effect of two same direction ter sites placed opposite oriC. The circular maps of pKZter_5′ter and pKZter_3′ter are shown as in Figure 1. The indicated plasmid (2.5 ng) was incubated in the RCR mixture at 30 °C for 3 h in the absence (0 nM) or presence (20 nM) of Tus. The product was analyzed by 0.5% TBE-agarose gel electrophoresis and SYBR Green I staining.

Figure 5

UvrD helicase rescues the detrimental effect of Tus in RCR. pPKOZ (−ter), pKZter_6 (Tus resistant), or pKZter_5 (Tus sensitive) was subjected to RCR propagation in the absence or presence of Tus as described in Figure 2A, except that the indicated concentration of UvrD was present in the reaction.

Figure 6

Effect of Cre-loxP system on the concatemer production in RCR. pUC_OriC300 (−loxP) or pUC_OLDT (+loxP) (0.05 ng) was incubated in the RCR mixture at 33 °C for 3 h in the absence (0 mU) or presence (5, 15, 50, or 150 mU) of Cre recombinase. The product was analyzed by 0.5% TBE-agarose gel electrophoresis and SYBR Green I staining. The ratio of concatemers to the sum of concatemers and supercoils is shown in a graph.