Structural
Insights into DNA Replication without Hydrogen
Bonds
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Abstract
The genetic alphabet
is composed of two base pairs, and the development
of a third, unnatural base pair would increase the genetic and chemical
potential of DNA. d<b>5SICS</b>-d<b>NaM</b> is one of
the most efficiently replicated unnatural base pairs identified to
date, but its pairing is mediated by only hydrophobic and packing
forces, and in free duplex DNA it forms a cross-strand intercalated
structure that makes its efficient replication difficult to understand.
Recent studies of the KlenTaq DNA polymerase revealed that the insertion
of d<b>5SICS</b>TP opposite d<b>NaM</b> proceeds via a
mutually induced-fit mechanism, where the presence of the triphosphate
induces the polymerase to form the catalytically competent closed
structure, which in turn induces the pairing nucleotides of the developing
unnatural base pair to adopt a planar Watson–Crick-like structure.
To understand the remaining steps of replication, we now report the
characterization of the prechemistry complexes corresponding to the
insertion of d<b>NaM</b>TP opposite d<b>5SICS</b>, as
well as multiple postchemistry complexes in which the already formed
unnatural base pair is positioned at the postinsertion site. Unlike
with the insertion of d5<b>SICS</b>TP opposite d<b>NaM</b>, addition of d<b>NaM</b>TP does not fully induce the formation
of the catalytically competent closed state. The data also reveal
that once synthesized and translocated to the postinsertion position,
the unnatural nucleobases again intercalate. Two modes of intercalation
are observed, depending on the nature of the flanking nucleotides,
and are each stabilized by different interactions with the polymerase,
and each appear to reduce the affinity with which the next correct
triphosphate binds. Thus, continued primer extension is limited by
deintercalation and rearrangements with the polymerase active site
that are required to populate the catalytically active, triphosphate
bound conformation