Accurate and Efficient
Bypass of 8,5′-Cyclopurine-2′-Deoxynucleosides
by Human and Yeast DNA Polymerase η
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Abstract
Reactive oxygen species (ROS), which can be produced
during normal
aerobic metabolism, can induce the formation of tandem DNA lesions,
including 8,5′-cyclo-2′-deoxyadenosine (cyclo-dA) and
8,5′-cyclo-2′-deoxyguanosine (cyclo-dG). Previous studies
have shown that cyclo-dA and cyclo-dG accumulate in cells and can
block mammalian RNA polymerase II and replicative DNA polymerases.
Here, we used primer extension and steady-state kinetic assays to
examine the efficiency and fidelity for polymerase η to insert
nucleotides opposite, and extend primer past, these cyclopurine lesions.
We found that <i>Saccharomyces cerevisiae</i> and human
polymerase η inserted 2′-deoxynucleotides opposite cyclo-dA,
cyclo-dG and their adjacent 5′ nucleosides at fidelities and
efficiencies that were similar to those of their respective undamaged
nucleosides. Moreover, the yeast enzyme exhibited similar processivity
in DNA synthesis on templates housing a cyclo-dA or cyclo-dG to those
carrying an unmodified dA or dG; the human polymerase, however, dissociated
from the primer–template complex after inserting one or two
additional nucleotides after the lesion. Pol η’s accurate
and efficient bypass of cyclo-dA and cyclo-dG indicates that this
polymerase is likely responsible for error-free bypass of these lesions,
whereas mutagenic bypass of these lesions may involve other translesion
synthesis DNA polymerases. Together, our results suggested that pol
η may have an additional function in cells, i.e., to alleviate
the cellular burden of endogenously induced DNA lesions, including
cyclo-dA and cyclo-dG