Replication of DNA-encoded information and its conversion into functional proteins are universal properties of life. In an effort toward the construction of a synthetic minimal cell, we implement here the DNA replication machinery of the Φ29 virus in a cell-free gene expression system. Amplification of a linear DNA template by self-encoded, de novo synthesized Φ29 proteins is demonstrated. Complete information transfer is confirmed as the copied DNA can serve as a functional template for gene expression, which can be seen as an autocatalytic DNA replication cycle. These results show how the central dogma of molecular biology can be reconstituted and form a cycle in vitro. Finally, coupled DNA replication and gene expression is compartmentalized inside phospholipid vesicles providing the chassis for evolving functions in a prospective synthetic cell relying on the extant biology.

Transcriptional activity in yeast strongly correlates with promoter occupancy by general factors such as TATA binding protein (TBP), TFIIA, and TFIIB, but not with occupancy by TBP-associated factors (TAFs). Thus, TBP exists in at least two transcriptionally active forms in vivo. The TAF-containing form corresponds to the TFIID complex, whereas the form lacking TAFs corresponds to TBP itself or to some other TBP complex. Heat shock treatment altered the relative utilization of these TBP forms, with TFIID being favored. Promoter-specific variations in the association of these distinct forms of TBP may explain why only some yeast genes require TFIID for transcriptional activity in vivo.

Bacteriophage φ29 DNA polymerase is a unique enzyme endowed with two distinctive properties, high processivity and faithful polymerization coupled to strand displacement, that have led to the development of protocols to achieve isothermal amplification of limiting amounts of both circular plasmids and genomic DNA. To enhance the amplification efficiency of φ29 DNA polymerase, we have constructed chimerical DNA polymerases by fusing DNA binding domains to the C terminus of the polymerase. The results show that the addition of Helix-hairpin-Helix [(HhH) ₂ ] domains increases DNA binding of the hybrid polymerases without hindering their replication rate. In addition, the chimerical DNA polymerases display an improved and faithful multiply primed DNA amplification proficiency on both circular plasmids and genomic DNA and are unique φ29 DNA polymerase variants with enhanced amplification performance. The reported chimerical DNA polymerases will contribute to make φ29 DNA polymerase-based amplification technologies one of the most powerful tools for genomics.

The DNA amplification performed by terminal protein-primed replication systems has not yet been developed for its general use to produce high amounts of DNA linked to terminal protein (TP). Here we present a method to amplify in vitro heterologous DNAs using the Φ29 DNA replication machinery and producing DNA with TP covalently attached to the 5′ end. The amplification requires four Φ29 proteins, DNA polymerase, TP, single-stranded DNA binding protein and double-stranded DNA binding protein (p6). The DNA to be amplified is inserted between two sequences that are the Φ29 DNA replication origins, consisting of 191 and 194 bp from the left and right ends of the phage genome, respectively. The replication origins do not need to have TP covalently attached beforehand to be functional in amplification and they can be joined to the DNA to be amplified by cloning or ligation. The facts that two functional origins were required at the ends of a linear template DNA and that the kinetics of DNA synthesis was very similar to that obtained using the TP-containing Φ29 genome as template support the proposal that genuine amplification is taking place. Amplification factors of 30-fold have been obtained. Possible applications of DNAs produced by this method are discussed.