Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination

Scientific Reports
Yoshihiro SakataniNorikazu Ichihashi

Abstract

A major challenge in constructing artificial cells is the establishment of a recursive genome replication system coupled with gene expression from the genome itself. One of the simplest schemes of recursive DNA replication is the rolling-circle replication of a circular DNA coupled with recombination. In this study, we attempted to develop a replication system based on this scheme using self-encoded phi29 DNA polymerase and externally supplied Cre recombinase. We first identified that DNA polymerization is significantly inhibited by Cre recombinase. To overcome this problem, we performed in vitro evolution and obtained an evolved circular DNA that can replicate efficiently in the presence of the recombinase. We also showed evidence that during replication of the evolved DNA, the circular DNA was reproduced through recombination by Cre recombinase. These results demonstrate that the evolved circular DNA can reproduce itself through gene expression of a self-encoded polymerase. This study provides a step forward in developing a simple recursive DNA replication system for use in an artificial cell.

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Citations

Apr 9, 2019·Annals of the New York Academy of Sciences·Norikazu Ichihashi
May 1, 2019·Chembiochem : a European Journal of Chemical Biology·Kensuke UedaNorikazu Ichihashi
Jul 26, 2019·Chembiochem : a European Journal of Chemical Biology·Daniel HürtgenVictor Sourjik
Jun 14, 2020·Protein Engineering, Design & Selection : PEDS·Yoshihiro SakataniNorikazu Ichihashi
Feb 2, 2019·Frontiers in Bioengineering and Biotechnology·Giordano RampioniPasquale Stano
Apr 9, 2020·Frontiers in Bioengineering and Biotechnology·Nadanai LaohakunakornSebastian J Maerkl
Jul 11, 2020·Advanced Biosystems·Daniel HürtgenVictor Sourjik
Jul 11, 2020·Advanced Biosystems·Daniel HürtgenPetra Schwille
Feb 16, 2020·Nature Communications·K LibicherH Mutschler
Jul 11, 2020·Advanced Biosystems·Kristian Le VayHannes Mutschler
Mar 18, 2021·Advanced Biology·Nathaniel J Gaut, Katarzyna P Adamala
Jul 18, 2020·ACS Synthetic Biology·Hiroki OkauchiNorikazu Ichihashi
Jun 27, 2020·Trends in Biotechnology·Alan Costello, Ahmed H Badran
Jul 28, 2021·Nature Communications·Lorenzo OliviJohn van der Oost
Aug 19, 2021·ACS Synthetic Biology·Hiroshi UenoHiroyuki Noji

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Methods Mentioned

BETA
electrophoresis
PCR

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