Reprograming the Replisome of a Semisynthetic Organism for the Expansion of the Genetic Alphabet

Semisynthetic organisms
(SSOs) created from Escherichia
coli can replicate a plasmid containing an unnatural base
pair (UBP) formed between the synthetic nucleosides dNaM and dTPT3 (dNaM-dTPT3) when
the corresponding unnatural triphosphates are imported via expression
of a nucleoside triphosphate transporter. The UBP can also be transcribed
and used to translate proteins containing unnatural amino acids. However,
UBPs are not well retained in all sequences, limiting the information
that can be encoded, and are invariably lost upon extended growth.
Here we explore the contributions of the E. coli DNA
replication and repair machinery to the propagation of DNA containing
dNaM-dTPT3 and show that replication by
DNA polymerase III, supplemented with the activity of polymerase II
and methyl-directed mismatch repair contribute to retention of the
UBP and that recombinational repair of stalled forks is responsible
for the majority of its loss. This work elucidates fundamental aspects
of how bacteria replicate DNA and we use this information to reprogram
the replisome of the SSO for increased UBP retention, which then allowed
for the first time the construction of SSOs harboring a UBP in their
chromosome.