Replication of E. coli duplex DNA in vitro. The separation of the DNA containing fractions of a lysate from the soluble enzymes and their complementation properties.
An E. coli lysate after being gently washed to remove soluble components, supports replicative DNA synthesis, if soluble proteins and the deoxyribonucleotide triphosphates are added. This DNA synthesis is dependent on ATP and on the presence of the gene products of the dnaB, dnaG, and polC (DNA polymerase III) genes. It continues at the replication forks preformed in vivo and "Okazaki fragments" are intermediate products of the reaction. Two different methods were used to prepare the washed DNA containing fraction. The one method involves washing of a cell lysate situated on a dialysis membrane. The other method involves DNAase treatment of a lysate and sedimentation of the degraded DNA through a glycerol gradient. Both washed preparation contain not only the DNA and the replication forks but also functional amounts of DNA polymerase III and of the dnaB gene product. Other factors, that are essential for replicative DNA synthesis, including the dnaG gene product, are washed out of the DNA containing preparations and the system is reconstituted by readdition of the soluble proteins.
D000255:Adenosine Triphosphate; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D004261:DNA Replication; D004269:DNA, Bacterial / Q000302:isolation & purification / Q000378:metabolism*; D003956:Dialysis; D004926:Escherichia coli
D004269:DNA, Bacterial; D000255:Adenosine Triphosphate; D004254:DNA Nucleotidyltransferases