Method of replicating or amplifying circular DNA

A method for replicating circular DNA in a cell-free system, comprising the following steps:

• (1) forming a reaction mixture of circular DNA as a template with a reaction solution comprising:
• more than one enzyme from a first enzyme group that catalyzes replication of circular DNA, wherein the first enzyme group comprises an enzyme having DnaA activity, one or more types of nucleoid protein, an enzyme or enzyme group having DNA gyrase activity, single-strand binding protein (SSB), an enzyme having DnaB-type helicase activity, an enzyme having DNA helicase loader activity, an enzyme having DNA primase activity, an enzyme having DNA clamp activity, and an enzyme or enzyme group having DNA polymerase III* activity,
• more than one enzyme from a second enzyme group that catalyzes an Okazaki fragment maturation and synthesizes two sister circular DNAs constituting a catenane, wherein the second enzyme group comprises an enzyme having DNA polymerase I activity and an enzyme having DNA ligase activity,
• one or more enzymes from a third enzyme group that catalyzes a separation of two sister circular DNAs, wherein the third enzyme group comprises an enzyme having topoisomerase IV activity and an enzyme having topoisomerase III activity;
• and
• a buffer, ATP, GTP, CTP, UTP, deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), and deoxythymidine triphosphate (dTTP), wherein the buffer is not water or a natural buffer;
• and
• (2) reacting the reaction mixture formed in step (1), wherein
• the circular DNA includes origin of chromosome (oriC) that can bind to an enzyme having DnaA activity, and further includes a pair of ter sequences that are each inserted outward with respect to oriC, and/or a nucleotide sequence recognized by a DNA multimer separation enzyme selected from a group consisting of Cre, XerCD, budding yeast (Saccharomyces verevisiae)-derived recombinant enzyme FLP, bacteriophage D6-derived recombinant enzyme DreO, Zygosacchromyces rouxii-derived recombinant enzyme R and a serine recombinant enzyme family consisting of Gin, yō, Tn3, and Hin, wherein
• when the circular DNA has the ter sequences, the reaction solution in step (1) further comprises a protein having an activity of inhibiting replication by binding to the ter sequences selected from a Tus protein or an RTP protein, and when the circular DNA has the nucleotide sequence recognized by the DNA multimer separation enzyme, the reaction solution in step (1) further comprises the DNA multimer separation enzyme, wherein each of the enzymes is a purified enzyme;
• and
• wherein the method does not require RNA-mediated transcription for initiation of replicating circular DNA.